Les neurotoxines botuliques (BoNTs) à l’origine du botulisme, présentent un intérêt thérapeutique majeur pour le traitement de diverses affections. La BoNT/B se lie aux neurones par l’intermédiaire de deux récepteurs membranaires : la synaptotagmine 2 (SYT2) et un poly-sialoganglioside de type GT1b. Dans une première partie, j’ai validé l’utilité d’un système permettant d’adresser vers les exosomes des protéines recombinantes complexes dont les récepteurs protéiques des BoNTs. Des tests de liaison de ligands spécifiques ont été réalisés avec succès. La BoNT/B se lie sur des exosomes exprimant la SYT2 en présence de GT1b avec une affinité (KD = 0.6 nM) comparable à celle obtenue avec des préparations de matériel natif. L’expression exosomale de protéines membranaires complexes représente ainsi une stratégie prometteuse pour des études pharmacologiques. Dans une seconde partie, j’ai montré que la BoNT/B se lie avec une faible affinité sur le GT1b dans des exosomes et que cette interaction n’est pas détectée dans un contexte cellulaire. En revanche la BoNT/B lie des exosomes exprimant la SYT2 avec une affinité de 40 nM ainsi que la SYT2 transfectée dans des cellules. La présence de GT1b dans les exosomes SYT2, induit un gain d’affinité d’un facteur 65. J’ai pu établir que la SYT2 lie directement le GT1b. La première lysine du domaine extra cellulaire de la SYT2 (K60) est directement impliquée dans cette interaction et sa mutation affecte la liaison de la BoNT/B sur le double recepteur SYT2/GT1b. À partir de ces résultats, nous proposons un nouveau modèle de liaison pour la BoNT/B, dans lequel la toxine interagit avec un co-récepteur SYT2/GT1b préalablement assemblé. / Botulinum neurotoxins (BoNTs) that are involved in botulism are also of major therapeutic interest for the treatment of several neurological conditions. To enter neurons, BoNT/B binds two membrane receptors: synaptotagmin 2 (SYT2) and GT1b, a poly-sialoganglioside. In a first part, I validated the utility of exosomes expressing recombinant forms of several complex membrane proteins, including protein receptors of BoNTs. Specific ligand-binding assays have been successfully performed. BoNT/B binds to exosomes expressing SYT2 and GT1b with affinity (KD = 0.6 nM) comparable to that found on native nerve-ending preparations. The exosomal expression of complex membrane proteins thus represents a promising technology for many pharmacological studies. In a second part of this study, I have shown that BoNT/B transiently binds with very weak affinity to GT1b in exosomal membranes and that this interaction is not detected in a cellular context. In contrast, BoNT/B binds to SYT2 on exosomes with an affinity of 40 nM as well as to transfected SYT2 in cultured cells in the absence of gangliosides. The presence of GT1b in SYT2 exosomes induces a 65-fold increase in BoNT/B affinity. I was able to establish that the extracellular juxta-membrane domain of SYT2 directly binds to the polar head of GT1b. The first lysine residue of SYT2 extracellular domain (K60) is involved in this interaction and its mutation affects the binding of BoNT/B to the dual receptor SYT2/GT1b. On the basis of these results, we propose a new model for synaptic terminal recognition by BoNT/B, in which the toxin interacts with a previously assembled SYT2/GT1b co-receptor.

Exosomes are microvesicles that are released from several different types of cells. Exosomes are thought to play an important role in functions such as immune regulation and coagulation; however their full functionality is not completely understood. Current research has started to explore their potential utilization in gene therapy and drug delivery. Their derivation from different proteins and RNA make them a versatile transport target in microbiology research. Although exosomes are being increasingly used in current research for gene therapy applications, the actual mechanism is unknown once the exosomes are taken into the cells. Using microfluidic channels, the entire process of exosome uptake can be imaged and monitored. The design of the microfluidic device allows for the manipulation of cellular flow and imitates the real flow of cells during exosome uptake and interaction. The microfluidic device is made from a mold using polydimethylsiloxane (PDMS) and the channels are coated with fibronectin for the cells to adhere to. The device is plasma bonded to a thin sheet of PDMS, incubated, and then left to cure. Because of its ability to grow quickly and efficiently in less-than-ideal conditions, hey ovarian cancer cells are used to seed the device. The hey cells are seeded at a density between five million and 10 million cells in the device, and fresh media is pumped through the device. The cells are left to adhere and proliferate for between 24 hours while fresh media is passed through the device in the 37 degrees C incubator. The hey cells are dyed using a DAPI fluorescent stain which causes the hey cells to illuminate blue fluorescence. Exosomes that are stained with PKH to illuminate green fluorescence are then seeded into the channels, and images are taken using confocal microscopy at several time points. The images showing blue fluorescent hey cells and green fluorescent exosomes are overlayed to show the exosomes uptake into the hey cells. Several images are taken across approximately a 20-minute time period to show the interaction between the exosomes and the cells in the channels.

Every year, millions of people in the world die from cardiovascular diseases. Most of thesevictims show no symptoms of disease for decades as the disease silently progresses, but maysuddenly become afflicted with a myocardial infarction or stroke. At present there is aregrettable lack of important disease markers that could help identify patients at risk withregard to secondary cardiovascular events. Biomarkers for this would of course be extremelyvaluable. If such markers could distinguish between patients with different responses totreatment and overall outcome, that would be of major added value as this potentially couldpave the way for differentiated preventive measures. In this study we show that specificproteins isolated from plasma-derived exosomes are indeed associated with an increased riskfor secondary cardiac events.

Urine contains exosomes originating from the circulation and all cells lining the urinary tract. Exosomes are a route of inter-cellular communication along the nephron potentially able to transfer of protein and/or RNA. It is not known whether this is a regulated process analogous to other cell-to-cell signalling systems. The aims of this study were to develop nanoparticle tracking analysis (NTA) as a technique to quantify exosomes in urine. Secondly, the hormonal regulation of exosome uptake in vitro and in vivo was investigated. Thirdly, exosome excretion in a central diabetes insipidus (DI) patient and a patient group after radiocontrast exposure was measured to investigate exosome excretion along the kidney in injury. Using the fluorescent capabilities of NTA, urinary exosomes were quantified in urine samples. NTA was able to detect changes in aquaporin 2 levels in vitro and in vivo. Storage conditions for human urinary exosomes were also optimised using NTA. A kidney cortical collecting duct cell line (CCDs) was used to model regulation of exosome uptake in vitro. CCDs were stimulated with desmopressin, a vasopressin analogue, and uptake of fluorescently-loaded or microRNA-loaded exosomes was measured. Desmopressin stimulated exosome uptake into collecting duct cells via V2 receptor stimulation. Intra-cellular uptake of exosomes was confirmed by microRNA specific mRNA down-regulation. Mechanistically, exosome uptake in response to desmopressin required cyclic AMP production, was mediated by clathrin-dependent endocytosis and was selective for exosomes from kidney tubular cells. In mice, fluorescently-loaded exosomes were systemically injected before and after administration of the V2 antagonist, tolvaptan, and urinary exosome excretion was measured. Basally, 2.5% of injected exosomes were recovered in urine; tolvaptan treatment resulted in a 5-fold increase. By combining antibodies to nephron segment-specific proteins with NTA we measured human urinary exosome excretion in central diabetes insipidus (DI) and after radiocontrast exposure (n=37). In DI, desmopressin reduced the excretion of exosomes derived from upstream glomerular and proximal tubule cells. In patients exposed to radiocontrast, urinary exosomes from the glomerulus were positively correlated with the tubular injury markers KIM- 1 and NGAL. These findings therefore show that tubular exosome uptake is a specific, hormonally regulated process that is reduced with injury. Physiologically, exosomes are a mechanism of inter-cellular communication; therapeutically, exosomes represent a novel vehicle by which RNA therapy could be targeted for the treatment of kidney disease.

E-selectin is an endothelial adhesion molecule important in the recruitment of leukocytes to the bone marrow and inflammatory tissues; moreover, it has also been implicated in cancer metastasis and as a critical mediator of chemoresistance. This study investigated the effects triggered by the binding of E-selectin on acute myeloid leukemia (AML) cell lines regarding the activation of significant signaling pathways and their impact on biological functions. We observed that upon treatment with a recombinant E-selectin construct on AML cells, there was an activation of the AKT/NF-κB pathways, which are known to be central regulators of many cellular processes, including survival and proliferation. We found that the E-selectin-mediated activation varied in rate and amplitude among AML cell lines spanning differentiation blockage at different stages. Furthermore, we found that E-selectin binding in HL-60 and KG1-a cells sensitized the cells to daunorubicin (DNR), a chemotherapy drug commonly used to treat AML. The observed chemosensitivity could be linked to the decrease of Aldo Keto Reductases (AKR) protein levels upon E-selectin treatment, which is known to metabolize DNR and reduce its cytotoxicity. Additionally, we explored the role of exosomes as a regulator of the generation of therapy resistance by examining the effects treatment with KG1-a derived exosomes, a cell line that exhibits higher chemoresistance compared to other AML cell lines, had on viability in HL-60 cells upon chemotherapy. We found that even in the absence of KG1-a cells, exosomes were sufficient to provide an increase in chemoresistance. Overall, these studies explore properties exerted by AML cells that could lead to further understanding of AML and thus the development of potential therapeutic targets to overcome challenges currently found in the treatment of this disease.

La Macroglobulinémie de Waldenström (MW) est un type de lymphome à cellules B de faible grade dont la pathogénèse est caractérisée par différents stades de progression. La MW asymptomatique n’occasionne pas de symptômes sévères chez les patients qui en sont atteints et nécessite une observation régulière mais pas de traitement. Elle peut évoluer en une forme symptomatique aux effets nocifs qui requiert un traitement par chimiothérapie. Le développement de la MW est généralement précédé par l’apparition d’un syndrome précurseur appelé Gammapathie Monoclonale de Signification Indéterminée à Immunoglobuline M (GMSI à IgM). Bien que les conséquences cliniques des formes asymptomatique et symptomatique de la maladie soient dramatiquement différentes pour les patients, au niveau cellulaire peu de différences permettant d’expliquer ce contraste ont été identifiées. Les mécanismes moléculaires de progression de la maladie restent donc majoritairement à élucider.Les exosomes sont des vésicules d’une taille de 40 à 100 nanomètres sécrétées par les cellules. Ils assurent le transport de protéines, de lipides et de molécules d’ARN ou d’ADN entre les cellules de l'organisme et constituent ainsi un moyen de communication intercellulaire. Ces propriétés permettent aux exosomes de conditionner le microenvironnement tumoral afin de le rendre favorable à la survie et la dissémination des cellules tumorales.Le but de cette étude est d’analyser le contenu des exosomes circulants chez les patients atteints de MW afin d’identifier des microARNs exprimés différentiellement en fonction du stade de progression de la maladie.Pour cela, les exosomes présents dans le sang circulant de patients à différents stades de la maladie (GMSI à IgM, MW asymptomatique et MW symptomatique) et d’individus sains ont été isolés. Le contenu en microARNs de ces exosomes a été analysé dans un premier temps par qRT-PCR puis par une technique utilisant la cytométrie de flux. Ces analyses ont permis d’identifier un groupe de microARNs dont l’expression corrèle directement ou inversement avec la progression de la maladie.En parallèle, l’ADN contenu dans les exosomes de certains de ces échantillons a également été extrait afin de détecter la fraction de variants mutés pour MYD88, un gène muté chez environ 90% des patients atteints de MW. Cette analyse permettra d’identifier une potentielle corrélation entre la progression de la maladie, la fraction de variants mutés de MYD88 et l’expression de certains microARNs dans les exosomes circulants. / Waldenström Macroglobulinemia (WM) is a low- grade B-cell lymphoma with a heterogenous clinical presentation. In patients with the asymptomatic form of the disease, no severe symptoms are observed and only monitoring is recommended. However, it can evolve into symptomatic WM in which case the patients will require treatment with chemotherapy .In many patients, the diagnosis is preceded with an asymptomatic precursor state of IgM monoclonal gammopathy of undetermined significance (MGUS). To date, the molecular mechanisms involved in the progression from asymptomatic WM to symptomatic WM remain to be elucidated.Exosomes are small vesicles secreted by cells with a size ranging from 40 to 100 nanometers that mediate the transfer of nucleic acids, proteins and lipids between distant cells in the organism. Exosomes enable communication between cells and the conditioning of distant tissues throughout the body.The goal of this study is to analyze the microRNA content of circulating exosomes in patients at different stages of WM progression to identify microRNAs which expression correlates with disease progression.Exosomes were isolated from the peripheral blood of healthy individuals and patients with WM at different stages (IgM MGUS, asymptomatic WM and symptomatic WM). The microRNA content of these exosomes was analyzed first by qRT-PCR and then by a technique involving flow cytometry. These analyses revealed a group of microRNAs which expression correlated directly or inversely with disease progression.In parallel, the DNA content of some of the exosomes was extracted in order to detect the fraction of mutated MYD88, a gene mutated in approximately 90% of patients with WM. This could potentially identify a correlation between disease progression, the fraction of mutated MYD88 and the expression of specific microRNAs in circulating exosomes.

Exosomes are small lipid membrane bound vesicles formed as part of the endosomal pathway and released into the extracellular space following fusion of late endosomes with the plasma membrane. Exosomes have been shown to have a variety of biological roles and may represent a novel source of disease biomarkers. The objectives of this project were to develop a panel of techniques for identifying exosomes in human urine then establish an in vitro model to determine whether exosomes change with cellular activation. We then used the techniques developed with human urine to determine whether human cerebrospinal fluid (CSF) contains exosomes and applied a mass spectrometry based approach to characterise the exosomal proteome. We used western blot for three exosomal markers (tsg101, CD24 and flotillin- 1), isopycnic centrifugation on a sucrose density gradient and direct visualisation using transmission electron microscopy (TEM) to verify the presence of exosomes. Using GeLC-MS/MS, 88 proteins were identified in the urinary exosomes. Several of these proteins could be linked to diseases and specific sections of the nephron. A murine cortical collecting duct cell line was used to model exosome release into the urine. Firstly, exosome release was verified using the approach developed in the urine. Stimulation of the cells with desmopressin caused an increase in the presence of aquaporin 2 in the exosomes. This increase reflected a similar change in the cells and occurred over a similar time course. This supports the hypothesis that the exosomes reflect the state of the kidney cells. In contrast, stimulation with cisplatin did not alter the presence of Fetuin-A, a proposed biomarker of cisplatin-induced acute kidney injury, in exosomes and this was consistent with no change in Fetuin- A expression in the cells. The released exosomes may act as mediators of communication to other cells. Following incubation of mCCD cells with AQP2 containing exosomes AQP2 in the cell lysate was increased indicating interaction between the cells and exosomes and potentially internalisation. Exosomes have been shown to be released by neuronal cells in vitro. We identified exosomes in the CSF of humans using western blot for known exosomal markers, density determination and direct visualisation with TEM and Immuno-TEM using an antibody specific for the exosomal marker flotillin-1. Label-free quantitative mass spectrometry was used to compare multiple CSF samples. On a whole protein analysis 86% of the proteins identified varied by less than 2-fold in comparison to the average across samples. On a tryptic peptide analysis 75% of the peptides identified varied by less than 2-fold in comparison with the average across samples. We have demonstrated exosomes are present in urine, CSF and mCCD cell conditioned media. In the mCCD cell derived exosomes we have demonstrated that following stimulation the proteome of the exosomes changes and that this change reflects the change seen in the cells. For the urinary and CSF exosomes we have characterised their proteomes using GeLC-MS/MS. These findings are consistent with the hypothesis that exosomes are a rich source of information, including biomarkers, on their cells of origin.

Le système immunitaire reconnait les cellules tumorales mais il est régulé par plusieurs facteurs tels les cellules T Régulatrices (Tregs). La galectine (Gal)-9 est une lectine aux propriétés immunosuppressives exprimée par les cellules cancéreuses et les cellules immunitaires dont les Tregs. Nous avons cherché à confirmer le rôle fonctionnel de la Gal-9 dans les Tregs. Puis nous avons testé la capacité d’un anticorps anti-Gal-9 (GalNab1) à bloquer les fonctions suppressives de la Gal-9 ou des Tregs et son effet anti-tumorale. Nous avons prouvé que les Tregs expriment et secrètent abondamment la Gal-9. GalNab1 antagonise l’effet de la Gal-9 recombinante (r) sur les PBMCs et inhibe les fonctions suppressives des Tregs. Le blocage de la rGal-9 en culture favorise la croissance des Th1 sans induire de cytotoxicité et bloque les fonctions des exosomesGal-9+ dérivés de Carcinome du Nasopharynx (CNP). In-vivo, dans un modèle de souris SCID humanisé, GalNab1 limite la croissance du CNP. Le CNP est associé au virus d’Epstein-Barr (EBV) dont il exprime plusieurs protéines. L’utilisation d’une stratégie de vaccination peptidique ciblant les lymphocytes TCD4+ Th1 est envisagée. Six peptides dérivés des antigènes de latence II d’EBV et promiscuous pour HLA-II ont été sélectionnés et assemblés en cocktail, dont la capacité à induire une sécrétion d’IFN-γ par les PBMCs a été validé. Des lignées Th1 spécifiques du cocktail présentent une forte capacité cytotoxique vis-à-vis de lignées de CNP tout en résistant aux effets des exosomes tumoraux autologues. In-vivo, le cocktail permet de maîtriser la croissance tumorale, et ex-vivo, de réactiver la réponse T mémoire chez les patients. / The immune system can recognize and eliminate cancer cells but is held back by inhibitory factors such as Regulatory T cells (Tregs). Gal-9 is a β-galactoside binding lectin with immunosuppressive capabilities expressed by cancer cells and immune cells including Tregs. NPC is a malignant epithelial cancer which is almost always associated with Epstein Bar Virus (EBV) and expresses several viral proteins. Numerous vaccines targeting different EBV peptides had limited success in clinical trials. First part: we aimed to confirm the role of Gal-9 in human Treg function. Then we tested the capabilities of an anti-human-Gal-9 antibody (mAb) to block Gal-9 suppressive function and its effect on Treg function and the anti-tumoral response. We proved that Gal-9 is expressed and secreted by Tregs at a high level. The mAb antagonized the function of recombinant rGal-9 on PBMCs. Moreover, the mAb inhibited the immuno-suppressive function of Tregs. Gal-9 blocking in PBMC culture promoted a Th1 response without inducing toxicity. We used the mAb to inhibit hNPC derived exosomes. In-vivo, the mAb limited the growth of hNPC tumors in humanized SCID mice. Second part: CD4+ T cell response is essential in managing NPC. The use of a CD4+ T cell response inducing peptide cocktail vaccination strategy was tested here. 6 HLA II promiscuous peptides derived from the 3 EBV latency II antigens were generated. These peptides induced IFNγ secretion by PBMCs. Generated peptide-specific CD4+ T cell lines showed highly cytotoxicity against NPC cell lines and resistance to hNPC exosomes. Invivo, the cocktail restrained tumor growth. Exvivo, it reactivated NPC patients’ memory T cells.

Background Breast cancer is the most commonly diagnosed cancer in women worldwide. The identification of breast cancer molecular biomarkers would provide a more accurate assessment of individual disease risks and prognosis. Exosomes, small extracellular vesicles, have been shown to contribute to various aspects of cancer development and progression. Within the last decade, the content of exosomes has been increasingly explored as a new source of potential biomarker molecules for early disease detection. Methods Exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were compared. Proteomic analysis was conducted using nano-liquid chromatography coupled to tandem mass spectrometry. The expression of proteins in MDA-MB-231cells was analyzed using label-free protein quantification methods. For the selection of potential biomarkers, the following criteria were used: (i) proteins must be unique to MDA-MB-231 cells when compared to MCF-10A cells, ii) localized on the membrane, (iii) abundant in breast cancer and (iii) are reported to increase in expression as the disease progresses. The presence of selected proteins on exosomes was verified using flow cytometry methods. Results In total, 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. The biomarker selection process identified three exosomal proteins (glucose transporter 1, glypican 1, and “disintegrin and metalloproteinase domain-containing protein 10”) as potential breast cancer biomarkers. The presence of these three proteins was validated using flow cytometry methods. The proteomics dataset was also rich in other interesting breast cancer proteins, such as 16 metastasis-associated proteins and two kinases. Conclusion We demonstrate that breast cancer exosomes are a rich source of protein biomarkers that may be beneficial for diagnosis and prognosis.

acase@tulane.edu / 1 / Adedoyin Johnson