<p>  </p> <p>Adoptive cell transfer of chimeric antigen receptors (CAR) T cells has successfully targeted hematological malignancies in human patients. However, unpredicted side effects experienced after injection of the CAR T cells suggests the need for an optimal predictive preclinical animal model. Dogs have intact immune systems and develop solid tumors spontaneously with similar morphology and genetics to humans. I hypothesize that generating CAR T cells for dogs will closely mimic human patients' outcomes, thus providing new understandings of the safety of this immunotherapy. In addition to the dog as a preclinical model, we propose using a universal CAR T cell to overcome various tumor-related immunosuppressive challenges and control the killing of the target cells. To achieve this, we established methods for activating and expanding canine T cells to a clinically relevant scale. Then, we expressed a second-generation anti-FITC-8-41BB-ζ CAR T cell via lentiviral transduction. In the presence of the correct low-molecular-weight bispecific adapter, we showed <em>in-vitro</em> CAR-mediated function. Our results proved that it is feasible to generate functional canine anti-FITC-8-BB-ζ CAR T cells for therapy.</p>

No description available.

La xénotransplantation, soit la transplantation de cellules, de tissus ou d'organes d'origine animale chez l'homme, est envisagée comme solution à la pénurie d'organes. Toutefois, cette technologie pourrait être à l'origine de nouvelles maladies. D'où la nécessité d'avoir des mesures visant tant la santé des animaux fournisseurs que la qualité et la sécurité des xénogreffons pour minimiser les risques de transmission de maladies de l'animal à l'homme, appelées xénozoonoses. L'objet de ce mémoire est de vérifier si les normes existantes au Canada et au Québec sont appropriées pour assurer la sécurité des receveurs et de la population. Nous avons d'abord examiné les normes susceptibles de s'appliquer à la surveillance et au contrôle de la santé des animaux fournisseurs. Ne visant que les maladies connues, elles ne répondent pas aux spécificités de la xénotransplantation. Quant aux xénogreffons, leur qualification pose problème: drogues ou instruments. Cette incertitude pourrait affecter l'uniformité des décisions relatives à leur qualité et à leur sécurité. Nous avons aussi étudié la Proposition d'une Norme canadienne pour la xénotransplantation. Cette dernière pourrait certes pallier la situation d'inadéquation de l'encadrement normatif existant au Canada. Une comparaison de cette norme canadienne avec les recommandations de l'OMS et les mesures en place aux ÉtatsUnis nous permet de suggérer comment la bonifier. Il ressort de notre étude que l'encadrement normatif canadien visant la sécurité des xénogreffons demeure à bâtir. Un élément essentiel à considérer dans son élaboration est la nécessité d'instaurer des systèmes de contrôle adéquats et compatibles à l'échelle planétaire. / Xenotransplantation that is, the transplantation of non-human animal cells, tissues and organs into humans, is considered a solution to the organ shortage problem. However, this technology could be the source of new diseases; therefore, it is necessary to have measures aimed at assuring the health of animal donors as well as the quality and safety of the xenografts in order to minimize the risks of disease transmission from animal to human, called xenozoonosis. The subject of this master's thesis is to verify if the standards that exist in Canada and in Quebec are appropriate to assure the safety of the recipients and the population. In the first place, we have examined the standards that could apply to the surveillance and control of the health of animal donors. These standards that apply to known diseases do not meet the specificities of xenotransplantation. As for the xenografts, their qualification is problematic: drugs or medical devices? This possibility of a double status could affect the uniformity of the decisions relative to their quality and safety. We have also studied the Proposed Canadian Standard for Xenotransplantation. The latter may resolve the unsuitability of normative standards that exists in Canada. A comparison of such a Canadian standard with the WHO recommendations and the measures in place in the United States allows us to recommend on how to improve it. Our study reveals that a Canadian normative standard that focuses on the security of the xenografts is still to be implemented. An essential element to be considered in its development, is the need to establish a worldwide compatible and suitable control system.

The shortage of human tissues and organs for allotransplantation can be overcome by xenotransplantation. As a source of organs, the miniature pig is convenient. However, the presence of pathogens transmissible to the recipients, especially porcine endogenous retrovirus (PERV), represents a threat for successfull xenotransplantation. Infectious PERVs contain three classes of envelope glycoprotein. Two classes, PERV-A and PERV-B are polytropic, they can infect human, pig and mink cells in vitro. PERV-C is evolutionary young, ecotropic isolate that can infect pig only. We previously detected a new full-lenght, but replication-defective PERV-A isolate dubbed (MAMBA) with high transcriptional activity in Large-White pig from a Czech breed. To support our results with PERV-MAMBA epigenetic regulation in pig tissues, in vitro DNA methylation essay was accomplished. Methylated or non-methylated reporter plasmids containing provirus 5' LTR were transfected into 293T cells and luciferase activity was measured. In both cases, methylated LTR decreased significantly expression of luciferase. Thus, PERV LTR-driven transcription is sensitive to DNA methylation. We also used PERV-A MAMBA provirus to study recombination between two pig endogenous retroviruses. We prepared 293T and BeWo cell clones harboring PERV-A...

Textbook accounts of vertebrate embryonic development have been based largely upon experiments on amphibian embryos, which have shown that the tissues of the trunk and tail are organised from distinct precursors that existed during gastrulation. In the mouse and chick, however, retrospective clonal analyses and transplantation experiments have demonstrated that the amniote body instead arises progressively from a population of axial precursors that are common to both the neural and mesodermal tissues of the trunk and tail. For this reason, they are known as neuro-mesodermal progenitors (NMps). Detailed studies of NMps have been precluded by their lack of a unique gene expression profile and the technical difficulties associated with isolating them from the embryo. Mouse embryonic stem cells (ESCs) provide the possibility of instead deriving them in vitro. ESCs have been used to model developmental processes, partly through large cellular aggregates known as embryoid bodies. These structures do not, however, resemble the axial organisation of the embryo and they develop in a disordered manner. This thesis presents a novel culture system of small, three-dimensional aggregates of ESCs (gastruloids) that can recreate the events of early post-implantation development, including axial elongation. Gastruloids are the first ESC-based model for axial elongation morphogenesis; this body of work characterises their development and identifies a candidate population of NMps within their elongating tissues. Additionally, this work establishes a xenotransplantation assay for testing the functional properties of in vitro-derived NMp populations in the chicken embryo and applies it to NMps from gastruloid cultures. The results of this assay show that gastruloids are a credible source of NMps in vitro and therefore offer a new experimental means to interrogate their properties. The use of gastruloids to recreate embryonic development has implications for basic research as a synthetic system and for the therapeutic derivation of other embryonic progenitors through bioengineering.

Introdução: Em mamíferos, a morte das células sensoriais (células ciliadas) da orelha interna acarreta perda auditiva neurossensorial permanente. Neste estudo, investigou-se se, após o transplante, as células-tronco (CT) da orelha interna de camundongos neonatos podem sobreviver e integrar nos giros basais das cócleas de cobaias submetidas à surdez induzida por neomicina. O potencial efeito do transplante celular na função auditiva também foi avaliada. Métodos: Oito cobaias foram submetidas à injeção de neomicina transtimpânica para indução de surdez. Após 7 dias, os animais foram aleatoriamente divididos em dois grupos. No grupo estudo (n=4), as CT da orelha interna de camundongos neonatos (meio de cultura com 105 CT LacZ+) foram transplantadas através de uma cocleostomia no giro basal de cobaias surdas. O grupo controle (n=4) recebeu somente meio de cultura. Após 14 dias do transplante, o efeito funcional foi avaliado por meio de pesquisa dos potenciais evocados auditivos e os animais foram sacrificados. A presença e a distribuição das CT transplantadas foram avaliadas por imunofluorescência dos cortes longitudinais das cócleas do grupo estudo. Testes não paramétricos foram usados para a análise estatística. Resultados: A ação da neomicina na cavidade timpânica proporcionou dano às células ciliadas e aumento significativo dos limiares auditivos antes do transplante celular. Não houve diferenças significantes dos limiares auditivos antes e após o transplante celular nas cobaias individualmente. Algumas CT transplantadas foram observadas em todas as escalas dos giros basais das cócleas lesadas, e parte destas células expressaram Miosina VIIA, um marcador de célula ciliada. Algumas CT transplantadas se integraram na membrana basilar da cóclea hospedeira. Nenhuma evidência de infiltração inflamatória nas cobaias transplantadas foi observada, apesar do uso de xenotransplante. Conclusões: Embora, após o transplante, os limiares auditivos não tenham sido alterados, os experimentos mostraram sobrevivência, migração, expressão de marcadores de células ciliadas e integração das CT transplantadas / Background: In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here we investigate whether, after transplantation, neonatal mouse inner ear stem cells (mIESCs) can survive and integrate into basal turns of neomycin-injured guinea pig cochleas. We also studied the potential effects of the cell transplantation on the auditory function. Methods: Eight adult guinea pigs were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. Study group (n=4) received transplantation to the scala tympani of 1 X 105 LacZ-positive mIESCs in culture medium. Control group (n=4) received culture medium only. Fourteen days after the transplantation, functional analyses were performed by auditory brainstem responses (ABR) and the animals were sacrificed. The presence and distribution of mIESCs were evaluated by immunohistochemistry of longitudinal sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis. Results: The intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs have been observed in all scalae of basal turns of the injured cochleas, and a proportion of those cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs integrated in the cochlear basilar membrane. There was no evidence of inflammatory infiltration in any of the guinea pigs. Conclusions: Although implanted mIESCs showed no obvious effect on auditory thresholds, our experiments showed survival, migration, expression of hair cell marker and integration of transplanted cells

Introdução: Em mamíferos, a morte das células sensoriais (células ciliadas) da orelha interna acarreta perda auditiva neurossensorial permanente. Neste estudo, investigou-se se, após o transplante, as células-tronco (CT) da orelha interna de camundongos neonatos podem sobreviver e integrar nos giros basais das cócleas de cobaias submetidas à surdez induzida por neomicina. O potencial efeito do transplante celular na função auditiva também foi avaliada. Métodos: Oito cobaias foram submetidas à injeção de neomicina transtimpânica para indução de surdez. Após 7 dias, os animais foram aleatoriamente divididos em dois grupos. No grupo estudo (n=4), as CT da orelha interna de camundongos neonatos (meio de cultura com 105 CT LacZ+) foram transplantadas através de uma cocleostomia no giro basal de cobaias surdas. O grupo controle (n=4) recebeu somente meio de cultura. Após 14 dias do transplante, o efeito funcional foi avaliado por meio de pesquisa dos potenciais evocados auditivos e os animais foram sacrificados. A presença e a distribuição das CT transplantadas foram avaliadas por imunofluorescência dos cortes longitudinais das cócleas do grupo estudo. Testes não paramétricos foram usados para a análise estatística. Resultados: A ação da neomicina na cavidade timpânica proporcionou dano às células ciliadas e aumento significativo dos limiares auditivos antes do transplante celular. Não houve diferenças significantes dos limiares auditivos antes e após o transplante celular nas cobaias individualmente. Algumas CT transplantadas foram observadas em todas as escalas dos giros basais das cócleas lesadas, e parte destas células expressaram Miosina VIIA, um marcador de célula ciliada. Algumas CT transplantadas se integraram na membrana basilar da cóclea hospedeira. Nenhuma evidência de infiltração inflamatória nas cobaias transplantadas foi observada, apesar do uso de xenotransplante. Conclusões: Embora, após o transplante, os limiares auditivos não tenham sido alterados, os experimentos mostraram sobrevivência, migração, expressão de marcadores de células ciliadas e integração das CT transplantadas / Background: In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here we investigate whether, after transplantation, neonatal mouse inner ear stem cells (mIESCs) can survive and integrate into basal turns of neomycin-injured guinea pig cochleas. We also studied the potential effects of the cell transplantation on the auditory function. Methods: Eight adult guinea pigs were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. Study group (n=4) received transplantation to the scala tympani of 1 X 105 LacZ-positive mIESCs in culture medium. Control group (n=4) received culture medium only. Fourteen days after the transplantation, functional analyses were performed by auditory brainstem responses (ABR) and the animals were sacrificed. The presence and distribution of mIESCs were evaluated by immunohistochemistry of longitudinal sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis. Results: The intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs have been observed in all scalae of basal turns of the injured cochleas, and a proportion of those cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs integrated in the cochlear basilar membrane. There was no evidence of inflammatory infiltration in any of the guinea pigs. Conclusions: Although implanted mIESCs showed no obvious effect on auditory thresholds, our experiments showed survival, migration, expression of hair cell marker and integration of transplanted cells

L'expression anormale de récepteurs hormonaux couplés aux protéines Gs (RCPG) a été mise en évidence dans des tumeurs cortico-surrénaliennes. Ces récepteurs contrôlent la sécrétion du cortisol, mais leur rôle dans la formation tumorale n'a pas été démontré. Nous avons utilisé un modèle de tumorigenèse cortico-surrénalienne permettant l'expression des gènes de deux récepteurs, le GIPR et le LHR, dont l'implication dans les manifestations cliniques d'hypercortisolisme a été bien établie. La formation de tissus hyperprolifératifs et hyperfonctionnels, d'aspect tumoral bénin, démontre pour la première fois que l'expression ectopique d'un seul gène de RCPGs (le GIPR ou le LHR) a un rôle initiateur de la tumorigenèse cortico-surrénalienne. Nous avons également caractérisé in vivo et in vitro un cas clinique de syndrome de Cushing sensible aux catécholamines, secondaire à une hyperplasie surrénalienne où nous avons démontré la surexpression du récepteur β2-adrénergique. La sécrétion de cortisol était aussi dépendante d'autres médiateurs tels que la 5-HT, l'AVP et l'ACTH sécrété localement de façon ectopique. En conclusion, l'expression d'un RCPG suffit pour induire la formation d'une tumeur bénigne cortico-surrénalienne. Des anormalités mixtes peuvent être présentes dans ce type de tumeur. Ce modèle permettra d'étudier les étapes de progression tumorale cortico-surrénalienne ainsi que l'effet d'autres types de GPCR et les alternatives thérapeutiques dans ce type de pathologie.

<p>In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. </p><p>Different <i>in vitro </i>systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans.</p><p>In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. </p><p>Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. </p><p>Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low <i>in vitro </i>but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.</p>

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.