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.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves : 325-355). Also available on the Internet.

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo. / A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin.

Les rétrovirus sont un enjeu de santé publique, aussi bien humaine qu'animale. La compréhension des déterminants structuraux sous-jacents à la fonction de leurs protéines constitue une étape essentielle dans le développement de stratégies antirétrovirales efficaces.Ce manuscrit porte sur l'étude des bases structurales des mécanismes moléculaires impliqués dans les fonctions clés des rétrovirus que sont i) la régulation de l'expression des protéines de rétrovirus complexes et ii) l'assemblage des particules virales, à travers l'étude de deux protéines rétrovirales d'intérêt thérapeutique : la protéine Tax du virus T-lymphotrope humain (HTLV) et la protéine de capside du virus de l'immunodéficience féline (FIV). L'étude structurale de ces deux protéines d'intérêt et la compréhension des mécanismes moléculaires nécessaires à leurs fonctions permettraient d'ouvrir la voie à la conception de nouvelles stratégies antirétrovirales.Malgré de nombreux tests d'expression et de purification, l'étude structurale de la protéine Tax du HTLV n'a pu être réalisée, en raison de son insolubité. Cependant, ce travail doctoral a permis de résoudre, pour la première fois, la structure cristallographique de la protéine de capside entière du FIV. Bien que cette dernière adopte un repliement similaire aux autres capsides rétrovirales dont la structure est connue, elle présente également des spécificités structurales dont les conséquences fonctionnelles seront discutées / Retroviruses are a major concern of public health in humans but also in animals. A better understanding of the structural determinants underlying the functions of retroviral proteins is a crucial step for the development of efficient antiretroviral therapies.This manuscript studies the structural basis of the molecular mechanisms implicated in key functions of retroviruses such as, i) the regulation of complex retroviruses protein expression and ii) the assembly of viral particles, through the study of two retroviral proteins of therapeutic interest: the human T-lymphotropic virus (HTLV) Tax protein and the feline immunodeficiency virus (FIV) capsid protein. The functional and structural studies of these two proteins and the understanding of the molecular mechanisms required for their functions will pave the way to the conception of new antiretroviral therapeutic strategies.Despite several expression and purification assays, no structural studies could be performed for the HLTV Tax protein. However, this study allowed the resolution of the first structure for the full-length FIV capsid protein by X-ray crystallography. Although the FIV capsid protein displays a standard a-helical topology like other retroviral CAs, it also harbors original features whose functional consequences will be discussed

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Les tumeurs rhabdoïdes (TR) constituent un rare cancer indifférencié du jeune enfant et du nourrisson, avec un âge médian au diagnostic de 20 mois. Ces tumeurs sont caractérisées par une inactivation biallélique du gène suppresseur de tumeur SMARCB1, un des membres du complexe SWI/SNF, acteur majeur du remodelage de la chromatine, sans autre altération génomique récurrente. Le pronostic des TR est péjoratif, le taux de survie globale atteignant 30% dans la plupart des séries, malgré des approches thérapeutiques conventionnelles particulièrement agressives. Les approches d’immunothérapies ont obtenu un succès certain dans certains cancers de l’adulte, et récentes analyses de l’infiltrat immun des cancers pédiatriques ne montrent pas un fort taux de tumeurs infiltrées à l’exception de rare types de cancers dont les TR intracrâniennes. Nous avons donc procédé à une analyse multimodale de l’infiltrat immun de cohortes de patients ainsi que d’un modèle de TR murines établi dans notre laboratoire. Nous avons identifié une forte proportion de tumeurs infiltrées dans certains sous-groupes de TR. Cet infiltrat était composé à la fois de cellules myéloïdes incluant des populations au phénotype immunosuppresseur, et lymphocytaires T notamment de phénotype résident mémoire caractérisées par une forte expansion clonale probablement spécifique d’un antigène tumoral. Nous avons identifié des cibles thérapeutiques communes aux tumeurs humaines et au modèle murin syngénique, et trouvé que cibler l’infiltrat lymphocytaire T ou myéloïde était susceptible d’induire une réponse tumorale complète avec induction d’une mémoire immunitaire, confirmant le caractère immunogénique des TR, et apportant de nouvelles stratégies thérapeutiques utiles en clinique. Enfin, nous avons identifié que les TR étaient le site d’une réexpression de rétrovirus endogènes, dépendante de celle de SMARCB1, avec activation des voies de l’interféron, apportant une base à une immunogénicité des TR issue du génome non codant. / Rhabdoid tumors (RT) are highly undifferentiated cancers occurring in infancy and early childhood, with a median age at diagnosis about 20 months. These tumors are characterized by the biallelic inactivation of SMARCB1 tumor suppressor gene, core member of the SWI/SNF complex, one major chromatin remodeling actor, in an otherwise highly stable genome. The prognosis of RT is dismal with overall survival hardly reaching 30% in most series, despite particularly aggressive conventional treatment. Immunotherapy approaches has gained a striking success within some adult cancer types and recent analyses of immune cell content of pediatric cancers don’t reveal a high rate of infiltrated tumors, except in few tumor types such as intracranial rhabdoid tumors. Then, we conducted a comprehensive analysis of the immune context of both human RT cohorts and a mouse RT model, including at single cell level. We identified a high recurrence of infiltrated tumors, in a RT-subgroup related manner, composed of both myeloid cells including cells with immune suppressive phenotypes, and T cells with notably a tissue resident memory phenotype demonstrating a high clonal expansion highly suggestive of immunogenicity. We identified common targetable immune populations between human and mouse RTs, and found that targeting both T and myeloid infiltrating cells was able to induce complete anti-tumor response with induced memory, confirming the immunogenic properties of RTs, and identifying new therapeutic strategies of clinical relevance. We finally identified that RTs were the site of SMARCB1-dependent endogenous retroviruses reexpression, with subsequent activation of interferon signaling, likely triggering the immune response in the context of RT, and providing a basis of non-coding genome-driven immunogenicity for these tumors.

APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.

Suite à une agression inflammatoire, telle que le choc septique, des brulures graves ou un traumatisme sévère, le système immunitaire répond par une modulation massive du transcriptome dans le sang. On propose d’explorer un autre répertoire que l’expression des gènes et de s’intéresser aux éléments répétés du génome, peu étudiés dans ces contextes, et plus particulièrement aux rétrovirus endogènes humains (HERV). Ils représentent plus de 8% du génome chez l’Homme. Certains sont exprimés dans des situations similaires à l’agression inflammatoire (cancer, maladies auto-immunes) et ont un impact sur la réponse immunitaire.Dans ce travail, nous cherchons à décrire et comprendre la contribution des HERV, au sein de la réponse immunitaire de l’hôte à l’agression inflammatoire. Pour cela, nous avons développé des méthodes et outils spécifiquement dédiés à la description du HERVome, au niveau génomique et transcriptomique. Nous montrons que les HERV sont exprimés dans le sang, modulés chez les patients, et que certains pourraient jouer un rôle sur l’expression de gènes de la réponse immunitaire situés à proximité. Nous évaluons également le polymorphisme de présence des HERV dans le génome de plus de deux mille individus répartis dans les populations humaines. On met en évidence que le polymorphisme HERV est globalement important, qu’il est lié à la population d’appartenance et que certains loci sont absents dans la majorité des génomes étudiés. Finalement, par différentes approches, nous identifions des associations entre gènes de la réponse immunitaire et HERV, suggérant que ces éléments peuvent jouer un rôle important dans la réponse de l’hôte à l’agression inflammatoire / Following inflammatory injury, like a septic shock, severe burn or important trauma, the immune system responds by a massive modulation of its transcriptome in the blood. We propose to explore another repertoire than gene expression and to focus on repeated elements, especially on HERVs. They represent more than 8% of the human genome. HERVs are expressed in similar settings (cancer or auto-immune diseases) and impact immune response. In this project, we describe and aim to better understand the HERV contribution in host immune response, following inflammatory aggression. To bring elements of response, we developed specifically dedicated tools to describe the HERVome, either at genomic or transcriptomic level. We show HERVs are expressed in blood in these settings, modulated in patients and could play a role on nearby gene expression. We also evaluate the polymorphism of presence of HERV loci on more than two thousands individuals, grouped into human populations. We show an important HERV polymorphism, that it is population-specific, and that some loci are absent in the majority of the analyzed genomes.Finally, with different approaches, we identify associations between immune-response genes and HERVs, suggesting these elements can play a role in host immune response following inflammatory aggressions

High grade aggressive lymphomas have high mortality. By their nature, more than 40% of patients die from these diseases even with the improved treatment strategies currently available for oncology patients. The characteristic feature is that they are functionally heterogeneous and therefore have different biological and molecular signatures which make it difficult for all groups to respond to same line of treatment. Based on the above, I set out to look at the impact of viral and cellular gene products on these groups of diseases: In chapter 3 I developed monoclonal antibodies against HERV‐K10. I subsequently investigated their expressions in aggressive lymphomas including Diffuse Large B‐cell lymphoma, Hodgkin’s lymphoma and Primary CNS lymphomas. I showed HERV‐K10 is expressed in cell lines of aggressive lymphomas, but not in paraffin‐embedded tissues. In chapter 4 I showed that the expression of ATM using immune‐histochemistry techniques in aggressive lymphomas does offer a guide to prognosis and treatment. Nearly 30% of Diffuse Large B‐cell lymphomas express ATM, 55% of Hodgkin’s lymphomas and more than 80% of Primary CNS lymphomas. I also showed there is a correlation of ATM expression and EBV‐driven aggressive lymphomas and that this has a poor prognostic significance. Chapter 5 analysed the results obtained by generating, validating and evaluating data base of DLBCL and PCNSL from a retrospective cohort over a 17‐year period. The results confirmed that prognostic indicators including ATM, S1PR2, Autotaxin and EBV using immuno‐histochemistry techniques help with categorising aggressive lymphomas into different prognostic groups and does influence future management. In summary, my results showed there is a critical place for immuno‐histochemistry techniques in convincingly helping understand the expressions of viral and cellular gene products in aggressive lymphomas and in contributing positively to their management.