Isolées à l'origine de la moelle osseuse , les cellules stromales mésenchymateuses (CSM) ont depuis été obtenues à partir de divers tissus fœtaux, postnataux et adipeux et font l'objet d'un nombre croissant d'essais cliniques en tant que traitement potentiel d’un large éventail de maladies dégénératives et de troubles autoimmuns. En effet, les CSM se sont révélées comme étant une alternative de premier plan pour la réparation tissulaire et la modulation immunitaire. Des recherches intensives ont visé à élucider leurs caractéristiques et les mécanismes par lesquelles elles provoquent leurs effets thérapeutiques. Plus précisément, dans le cadre de la transplantation de cellules souches hématopoïétiques (CSH) allogéniques chez des patients pédiatriques atteints de leucémie, les CSM ont été utilisées pour favoriser la greffe et/ou pour induire une immunosuppression dans la maladie du greffon contre l’hôte (GVH), une complication fréquente et mortelle de la greffe pour laquelle il n’y a que très peu à offrir aux patients qui en sont touchés. En effet, la première indication thérapeutique d’un produit à base de CSM approuvée par Santé Canada a été indiquée pour la gestion de la GVH chez des patients pédiatriques ayant une maladie résistante aux traitements de première intention. Traduire la recherche fondamentale en applications cliniques nécessite un approvisionnement régulier en CSM viables et préalablement évaluées quant à leur sécurité et leurs propriétés fonctionnelles. La cryopréservation des CSM est l’élément clé qui permet de répondre à ces exigences. Suivant l'exemple des CSH, qui ont été cryoconservées et transplantées avec succès ultérieurement, les CSM ont donc été entreposées et décongelées selon les besoins des études cliniques de phase III pour le traitement de la GVH réfractaire aux traitements classiques. Ces études ayant obtenu des résultats mitigés et contrastants avec les résultats obtenus à partir de cellules fraîches dans des études cliniques de phases I et II, la cryopréservation a rapidement été reconnue coupable de leur échec. Une quantité remarquable de recherches et de ressources ont ensuite été consacrées à l'optimisation des protocoles, à la composition des milieux de congélation, aux dispositifs de refroidissement et aux conteneurs de stockage, ainsi qu'au développement de bonnes pratiques de fabrication afin de garantir que les CSM conservent leurs caractéristiques thérapeutiques après la cryoconservation et qu’elles soient sécuritaires pour une utilisation clinique. L’objectif principal de cette thèse était de clarifier l’impact de la cryopréservation sur les propriétés immunosuppressives des CSM. Alors que les études publiées sur le sujet faisaient, d’une façon assez équilibrée, l’état d’une réussite ou d’un échec à la cryopréservation des CSM en lien avec la conservation de leurs différentes fonctions in vitro et/ou in vivo dans des modèles animaux, mes travaux ont permis de revisiter l'impact de la cryoconservation sur les CSM en démontrant que des épisodes de réchauffement cellulaire survenant après leur congélation, plutôt que la cryoconservation en elle-même, étaient responsables de leurs altérations fonctionnelles. Si des précautions ne sont pas prises pour éviter le réchauffement des échantillons lors du processus d’entreposage dans l’azote liquide, ceux-ci vont subir une iii importante variation de température et ce, de façon étonnamment rapide et dommageable. Mes travaux ont démontré que l’inhibition de la prolifération lymphocytaire par les CSM diminuait drastiquement et de façon proportionnelle au réchauffement subi par les CSM pendant l’entreposage des produits cellulaires. Étonnamment, la viabilité des CSM n’était pas significativement diminuée par ces variations de température alors qu’elle est depuis toujours la principale mesure utilisée pour quantifier l’intégrité cellulaire postdécongélation. Dans une perspective de pouvoir prédire la présence d’une atteinte fonctionnelle des CSM induite par la cryopréservation, la suite de mes travaux a permis d’identifier la capacité d’adhésion des CSM comme étant un marqueur rapidement et facilement mesurable en laboratoire, mais surtout étant significativement liée à la capacité des CSM à inhiber une réponse lymphocytaire, laquelle est au cœur des symptômes de la GVH. Une grande rigueur pendant tout le processus de fabrication et de congélation des CSM ainsi que la génération de données reproductibles et fiables sont indispensables à l’obtention de preuves scientifiques de l'efficacité et de la qualité de cette thérapie cellulaire à grand potentiel dans le traitement des maladies auto-immunes. / Originally isolated from the bone marrow, mesenchymal stromal cells (MSC) have since been obtained from various foetal, postnatal and adipose tissues and are the subject of an increasing number of clinical trials as a potential treatment for a wide range of degenerative diseases and autoimmune disorders. Indeed, MSC have proven to be a leading alternative for tissue repair and immune modulation. Intensive research has aimed to elucidate their characteristics and the mechanisms by which they induce therapeutic effects. More specifically, in the context of transplantation of allogeneic hematopoietic stem cells (HSC) in pediatric patients with leukemia, MSC have been used to promote transplantation and/or to induce immunosuppression in graft-versus-host disease (GVH), a frequent and fatal complication of transplantation for which there is very little to offer to patients who are affected. The first therapeutic indication for a MSC-based product approved by Health Canada was indicated for the management of GVH in pediatric patients with a resistant to first-line treatments disease. Translating basic research into clinical applications requires a regular supply of viable MSC that have been previously assessed for their safety and functional properties. Cryopreservation of MSC is the key to meeting these requirements. Following the example of HSC, which were cryopreserved and successfully transplanted later, MSC were therefore stored and thawed as needed in phase III clinical studies for the treatment of GVH refractory to conventional treatments. These studies having obtained mixed results and contrasting with the results obtained from fresh cells in clinical studies of phases I and II, cryopreservation was quickly found guilty of their failure. A remarkable amount of researches and resources were then devoted to the optimization of protocols, the composition of freezing media, cooling devices and storage containers, as well as the development of good manufacturing practices in order to guarantee that MSC retain their therapeutic characteristics after cryopreservation and that they are safe for clinical use. The main objective of this thesis was to clarify the impact of cryopreservation on the immunosuppressive properties of MSC. While the studies published on the subject made, in a fairly balanced way, the state of a success or a failure in cryopreservation of MSC in connection with the conservation of their different functions in vitro and/or in vivo in animal models, results presented in this thesis have allowed us to revisit the impact of cryopreservation on MSC by demonstrating that episodes of cell warming occurring after their freezing, rather than cryopreservation itself, were responsible for their functional alterations. If precautions are not taken to prevent the samples from heating up during the liquid nitrogen storage process, they will undergo a significant temperature variation, surprisingly quickly and very damagingly. My work has shown that the inhibition of lymphocyte proliferation by MSC decreases drastically and in proportion to the warming experienced by MSC during storage of cellular products. Surprisingly, the viability of MSC was not significantly reduced by these temperature variations, whereas it has always been the main measure used to quantify post-thaw cell integrity. In a perspective of being able to predict v the presence of a functional impairment of MSC induced by cryopreservation, the rest of my work then made it possible to identify the adhesion capacity of MSC as being a marker quickly and easily assessable in the laboratory, but especially being significantly linked to the ability of MSC to inhibit a lymphocyte response, which is at the heart of GVH symptoms. Great rigor throughout the manufacturing and freezing process of MSC as well as the generation of reproducible and reliable data are essential to obtain scientific evidences of the efficacy and quality of this cell therapy with very high potential in the treatment of autoimmune diseases

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Certain strains of mice, e.g. C57BL/6, are highly resistant to serious infections with the protozoan pathogen, Leishmania major, whereas other strains, e.g. BALB/c, are not. It has beenproposed that interferon gamma (IFN-y) is one of the most critical lymphokines produced in a protective response to these intracellular pathogens. IFN-y has been classified as a Thi lymphokine produced by the Thl subset of T lymphocytes which not only activates macrophages to kill the protozoa but also helps regulates the immune system overall to form a lasting immunity to the microorganism (4,19). Mice susceptible to L. major arethought to produce inadequate amounts of IFN-y and instead produce an excessive amount of a Th2 lymphokine, IL-4, produced by Th2 T cells. (6) We have previously found that prophylactic treatment with cyclosporine A (CsA), a T cell specific immunosuppressant, reduces the susceptibility of the BALB/c to L. major by either preventing disease entirely or delaying itsdevelopment significantly (19). In this murine model, it may be that CsA causes a switch from the production of the less protective Th2 lymphokines to the more protective Thl lymphokines. In order to test this hypothesis we examined the IFN-y produced by lymph node and spleen cells over time after infection in three groups of mice: C57BL/6, BALB/c and cyclosporine- protected BALB/c. Interestingly, cells taken from all three groups of mice were able to secrete IFN-y in vitro in response to co-culture with Leishmania, antigens. The pattern of secretion over time, however, varied and may indicate a significant difference in the animals' response to the pathogen. In addition to this work, we also examined the ability of another immunosuppressant, FK506, which is very similar in action to but much less toxic than CsA, to induce enhanced resistence to L. major. FK506 also appears to be effective in reversing the susceptibility of the BALB/c mice towards this pathogen with much less apparent toxicity. / Department of Biology

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Myeloid-derived suppressor cells (MDSC), emerging during tumor growth or chronic inflammation play a critical role in regulating T cell function. However, mechanisms governing the generation of these cells remain unclear, and need to be further defined. Using a DSS-induced colitis and recovery model, we characterized the dynamic changes within myeloid compartments and the emergence of MDSC during active and resolution phases of inflammation. We show that the immature myeloid compartment expands in bone marrow (BM) specifically at the resolution phase of inflammation during colitis transition to recovery. Additionally, we found enhanced levels of IL-17 in the serum of colitis mice tightly correlates with expansion of the IMC compartment, and is likely the factor responsible for expansion of these cells. Our study also determined that the expanded population of myeloid cells underwent a functional reprogramming event. In particular, two major functional changes occurred when colitic mice were allowed to recover: 1) CD11b+Gr-1+ myeloid cells in bone marrow and spleen acquired T cell suppressive functions, and 2) acquired the ability to enter into circulation from BM, confirming previously reported characteristics of MDSC. Additionally, we determined that acquired migratory capability in the low density myeloid cells isolated from resolution time points was due to enhanced surface expression of chemokine receptor CXCR2. Furthermore, we determined that after mobilization of MDSC from the bone marrow, these cells collected in the T cell-rich spleens, where they effectively functioned to suppress T cell proliferation. Through these acquired functions, our study determines a protective role for MDSC during the recovery phase of post-acute inflammation during persistent DSS-induced colitis.

Resumo: A resposta imune na leishmaniose pode resultar em uma polarização da subpopulação dos linfócitos T, levando a um fenótipo celular distinto, que resultam em proteção imune ou exacerbação da doença. As leishmânias persistem no organismo tanto em infecções assintomáticas como após o tratamento, representando risco em condições de imunossupressão. O objetivo deste trabalho foi avaliar o efeito da infecção e da imunossupressão com dexametasona associada à pentoxifilina, sobre o peso dos animais, peso do baço, carga parasitária do baço e do fígado, e da imunopatologia quanto à produção de IFN-γ, IL-10, IL-4 e IL-2 em cultura de células esplênicas de camundongos Balb/c infectados com Leishmania chagasi. A infecção não alterou o ganho de peso dos animais, mas sim o peso e o tamanho do baço. A imunossupressão utilizando a dexametasona associada à pentoxifilina afetou tanto ganho de peso, quanto o peso e o tamanho do baço, de animais infectados e não infectados. A imunossupressão não alterou significativamente o curso da carga parasitária, tanto do baço, como do fígado. A dexametasona e a pentoxifilina afetaram a produção das citocinas estudadas, mas não direcionaram o perfil de resposta Th1/Th2 nos animais infectados. / Abstract: The immune response in leishmaniasis can result in a polarization of a subpopulation of T lymphocytes, leading to a different cell phenotype, resulting in immune protection or exacerbation of the disease. Leishmanias persist in the body both in asymptomatic infections and after the treatment, representing risk in terms of immunosuppression. The objective of this study was to evaluate the effect of infection and immunosuppression with dexamethasone associated with pentoxifylline on the weight of the animals, weight of the spleen, the parasitic load in the spleen and liver, and immunopathology on the production of IFN-γ, IL - 10, IL-4, and IL-2 in spleen cell culture of Balb/c mice infected with Leishmania chagasi. The infection did not alter the animals' weight gain, but the weight and size of the spleen increased. The immunosuppression using dexamethasone associated with pentoxifylline affected both weight gains, as the weight and size of the spleen of infected and not infected animals. The immunosuppression did not alter significantly the course of the parasite burden of the spleen and the liver. Dexamethasone and pentoxifylline affected the studied cytokines production, but not influenced the profile of Th1/Th2 response in infected animals. / Orientador: Helio Langoni / Coorientador: Luciane Alarcão Dias-Melício / Banca: Angela Maria Victoriano de Campos / Banca: Lisiane de Almeida Martins / Mestre

It is well established that females are more immunocompetent than males as evidenced by higher humoral antibody titers, lowered susceptibility to infection, and more efficient graft rejection. Furthermore, females also exhibit a much higher incidence of autoimmune disease. These observations have led investigators to believe that the male hormonal environment may play a key role in the regulation of immune response. For this reason, this study is concerned with the expression of autoimmunity and of immune function in the mouse. This study included the New Zealand Black (NZB) mouse strain, as an animal model for human SLE, as well as normal DBA/2 and Balb/c strains. Animals were administered testosterone via subcutaneous implants in silastic tubing or by injection. Mice used were intact females, intact males and castrated males. Animals were otherwise untreated or had been exposed to a sublethal dose (400-550 rads) of irradiation. Target organ weight changes, immune capacity and peripheral blood picture changes were measured.

Typescript (photocopy) / 199 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--Dept. of Medicine, University of Adelaide, 1981

Bibliography: leaves 234-314. Examines the role of vascular endothelial cells in inflammation with particular reference to their participation in the immune response directed against a vascularised allograft (kidney)

Typescript (photocopy)