published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

Damage to the adult mammalian central nervous system (CNS), either by traumatic injury or disease, usually results in permanent sensory and/or motor deficits. Regeneration of neural circuits is limited both by the lack of growthpromoting molecules and by the presence of growth-inhibitory molecules in the mature brain and spinal cord. The research described here examines the therapeutic potential of viral vectors and neuronal transplants to reconstruct damaged neural pathways in the CNS. Experimental neural transplantation techniques often fall short of expectations because of limited transplant survival and insufficient neurite outgrowth to repair connections and induce behavioral recovery. These shortcomings are addressed in the current studies by virus-mediated expression of cell-specific neurotrophic and guidance molecules in the host brain prior to cell transplantation. The initial proof-of-principle studies show that viral vectors can be used to create axon-guidance pathways in the adult mammalian brain. With such pathways in place, subsequent transplantation of neurons leads to longdistance, targeted outgrowth of neurites. Application of this technique to a rat model of Parkinsons disease demonstrates that circuit reconstruction leads to functional recovery. For this study, rats were lesioned on one side of their brain with 6-hydroxydopamine to produce a hemiparkinsonian state. The motor deficit was confirmed by amphetamine-induced rotation testing and spontaneous motor asymmetry testing. The rats were then divided into experimental groups to receive lentivirus injections along a path between the substantia nigra (SN) and the striatum to express glial cell-line derived neurotrophic factor (GDNF), GDNF family receptor alpha-1 (GFR1), netrin-1 or green fluorescent protein (GFP, control). One group received combination injections of lenti-GDNF and lenti-GFR1. One week after virus injections, animals received transplants of embryonic midbrain dopaminergic neurons into their SNs. They were tested for motor asymmetry every two weeks for a total of eight weeks and then brain tissue was harvested for immunohistochemical analysis. Results demonstrate that virus-induced expression of GDNF and GFR1 supports growth of dopaminergic fibers from cells transplanted into the SN all the way to the striatum, and these animals have a significant reduction in both drug-induced and spontaneous motor asymmetry.

La transplantation d’hépatocytes est une nouvelle approche thérapeutique pour le traitement des maladies métaboliques. Elle peut être proposée en alternative à la transplantation de foie entier ou, à tout le moins, en attente de celle-ci chez les patients instables, à risque de décompensation métabolique. Les essais cliniques effectués chez 9 patients aux cliniques St Luc ainsi que ceux publiés dans la littérature démontrent l’intérêt de la transplantation de cellules hépatiques à court et moyen terme. La qualité de la suspension cellulaire transplantée reste le premier facteur limitant pour le développement clinique de la technique. La cryopréservation reste le moyen le plus approprié pour la conservation à long terme des cellules. Elle permet de constituer une banque de cellules pouvant être utilisées à tout moment. Nous avons d’abord analysé les protocoles de cryopréservation décrits dans la litérature, ainsi que leurs limites tant au niveau de la préservation de la qualité cellulaire après décongélation in vitro qu’après transplantation in vivo. Dans ce travail, nous avons démontré l’intérêt d’utiliser des cellules cryopréservées/décongelées, afin de stabiliser des patients atteints de maladies du cycle de l’urée, avant la greffe de foie entier. Les tests de contrôle de qualité effectués sur ces cellules ont cependant montré une altération aux niveaux biochimique et cellulaire, après décongélation. Nous avons ainsi démontré une chute des concentrations intracellulaires d’ATP, signe d’une atteinte mitochondriale. Nos travaux ont également permis de mettre en évidence une diminution de la consommation d’oxygène des hépatocytes en suspension, due plus particulièrement à une atteinte du complexe 1 de la chaîne respiratoire. Cette atteinte mitochondriale peut déjà être observée après l’incubation de la suspension cellulaire à –20°C. Aux alentours de cette température critique se fait le passage de l’état aqueux à l’état cristallin suggérant que les dégâts mitochondriaux observés sont dès lors vraisembablement dus à la formation de glace intracellulaire durant le processus de cryopréservation ou de décongélation. Diverses tentatives visant à améliorer les paramètres mitochondriaux affectés par le processus de congélation/décongélation par l’addition d’agents protecteurs du complexe 1 (Bilobalide), d’ inhibiteurs du pore de transition de perméabilité (Ciclosporine A), d’ anti-oxydants ou encore de solutions hyperosmotiques à la solution de cryopréservation, n’ont pas permis d’améliorer la qualité cellulaire. Le tri de sous-types de populations hépatocytaires ou l’isolement de foies hépatectomisés n’ont pas permis de révéler de différences de capacité de résistance à la cryopréservation. Toujours dans le but d’améliorer le rendement de la transplantation d’hépatocytes et d’augmenter l’efficacité d’implantation dans le parenchyme receveur, nous avons démontré dans la deuxième partie de la thèse la capacité des hépatocytes isolés (fraîchement isolés ou cryopréservés/décongelés) à induire un phénomène de coagulation dépendant du facteur tissulaire. Cette activité pro-coagulante, inhibée in vitro par lea N-acetyl-L-cystéine, pourrait être le point de départ d’une réaction inflammatoire aspécifique influençant ainsi la réussite de la transplantation cellulaire. En conclusion, nous proposons dans ce travail différentes stratégies en vue de l’amélioration du rendement de la thérapie cellulaire. La vitrification, autre technique de cryopréservation, permettrait d’éviter la formation d’eau intracellulaire. Enfin la modulation de l’activité pro-coagulante par la N-acetyl-L-cystéine, due à la transplantation cellulaire, constitue une piste intéressante pour essayer d’améliorer l’implantation des cellules transplantées et ainsi le rendement de la greffe. / Liver cell transplantation provides clinical benefit to patients with congenital metabolic abnormalities and currently represents an alternative to orthotopic liver transplantation or at least an interim measure for unstable patients awaiting transplantation. Our team and others have already demonstrated that transplanted hepatocytes can achieve metabolic control in the short or medium term. The quality of transplanted cells remains the first limiting factor for the success of liver cell transplantation. Because the use of freshly isolated cells is restricted by contemporary organ donation, cryopreservation remains necessary for long-term storage and permanent availability of the cells. In this thesis, we have first reviewed and discussed established hepatocyte cryopreservation protocols, especially the cooling procedure, and have focussed on the in vitro and in vivo assays used for the evaluation of post-thawing hepatocyte quality. Amongst 9 cell transplanted patients in our center, several received exclusively or predominantly cryopreserved/thawed hepatocytes. We demonstrated post-transplantation benefits of using these cells in control patients with congentital abnormalities in the urea cycle, particularly with respect to clear evidence of cell engraftment and de novo appearance of enzyme activity. However, despite these clinical benefits, we found an in vitro relationship between the low post-thawing quality of cryopreserved /thawed hepatocytes and an alteration in their mitochondrial function. This post-thawing mitochondrial damage was already evident after the first −20°C cryopreservation step of our protocol, suggesting it occurrs early in the process, around the nucleation point, by intracellular ice formation. Cellular impairment could therefore be possibly explained by mechanical alteration of mitochondria due to water crystallisation during the cryopreservation process or thawing procedure. We also observed a poor efficacy of cryopreserved/thawed hepatocytes (as compared to freshly isolated cells) when used liver engraftment in two mice transplantation models. The marked reductions in intracellular ATP concentrations and the decreases in oxygen consumption by hepatocytes were therefore used as markers for the evaluation of the effects of several compounds such as bilobalide, hyperosmotic or anti-oxidant molecules, pore transition permeability inhibitors, and for the evaluation of the resistance of selected hepatocyte subtypes to cryopreservation protocols. We also demonstrated that isolated hepatocytes exert tissue factor-dependent pro-coagulant activity, which may contribute to the early loss of infused cells. We observed that the addition of N-acetyl-L-cysteine to hepatocyte suspensions inhibits coagulation activation. In conclusion, this work has identified several ways to improve the clinical benefit of liver cell transplantation, including new cryopreservation strategies, such as vitrification. In addition, modulation of the pro-coagulant activity induced by cell infusion with N-acetyl-L-cysteine might beneficially enhance cell engraftment.

No description available.

No description available.

Samples from 93 of 115 consecutive cadaveric renal transplants were selected to define polymorphisms in both IFN-γ and IL-10. A 12 CA repeat IFN-γ polymorphic allele was found in 73 patients (70 in patients analysed further). This polymorphism was associated with high IFN-γ production in vitro. According to the presence or not of the 12 CA repeat allele patients were separated in high and low producer genotype groups. The incidence of acute rejection was 54.3% in this high IFN-γ genotype group, contrasting with 44.4% in the low IFN-γ. Requirement for ATG therapy was greater in the high IFN-γ group (odds ratio [OR]=2.5). Among HLA-DR-mismatched patients, IFN-γ high producer genotype was more strongly associated with rejection (OR=1.6). In the cyclosporine monotherapy subgroup, 11 out of 14 patients with IFN-γ high genotype (78%) had acute rejection (OR=2.88, p=0.09). Graft survival was similar between the two IFN-γ groups. When the analysis was controlled for the presence of delayed graft function, 40.5% of the high IFN-γ genotype patients had serum creatinine levels above 200 micromoles/L contrasting with only 14.3% of the low IFN-γ genotype recipients at 5 years after transplantation (p=0.05). In a regression model of creatinine at 1 year the significant variables were the presence of DGF, donor age greater than 50, greater than two rejection episodes, DR mismatch, donor female to male recipient sex, IL-10 high genotype, and IFN-γ high genotype. Conclusion: The 12 CA repeat IFN-γ polymorphic allele is associated with high IFN-γ production. We have shown that this high producer genotype for IFN-γ influences acute rejection in kidney transplantation, particularly in high-risk groups; it is also associated with worse long-term graft function.

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

Varje år genomförs omkring 700 organtransplantationer i Sverige. Patienter som genomgått en organtransplantation har en ständig risk för avstötning. Avstötning innebär att det transplanterade organet stöts bort och slutar fungera på grund av kroppens immunförsvar. Oavsett var transplantationen utförs så följs patienterna upp på sin hemort. Därför är det av stor vikt att som sjuksköterska inneha kunskaper om patienters upplevelser om avstötning. Syftet med litteraturstudien var därför att beskriva patienters upplevelser om avstötning efter en organtransplantation. För att uppnå syftet med studien utfördes en litteraturstudie, där fyra kategorier framstod i resultatet: Det ständiga hotet, En oönskad livssituation, Fångad i behandlingsregimen och Att finna styrka i vardagen. Avstötning är en upplevelse av ett ständigt hot som även kan medföra att patienter upplever ett emotionellt trauma. Patienterna känner sig tvungna att följa den utvalda behandlingsregimen för att undvika en avstötning. Efter en upplevd avstötning behöver patienterna använda sig av olika strategier för att kunna hantera vardagen. Ytterligare forskning om ämnet behövs för att kunna möta denna patientgrupps vårdbehov. / Each year about 700 organ transplants are performed in Sweden. Patients who have had an organ transplant are at constant risk of graft rejection. Graft rejection means that the transplanted organ is rejected and stops functioning because of the body’s immune system. Regardless of where the transplant is performed the patients are followed up at their domicile. Therefore it is of great importance that the nurse has knowledge about patients’ experiences in conjunction with graft rejection. The purpose of this study was to describe patients' experiences of graft rejection after organ transplantation. To achieve the purpose of this study a literature review was conducted, in which four categories emerged in the results: The constant threat, An undesirable situation in life, Caught in the treatment regimen, and To find strength in everyday life. Rejection is an experience of a constant threat that may cause patients to experience an emotional trauma. Patients feel obliged to follow the chosen treatment regimen to prevent rejection. After an experienced rejection, patients need to use different strategies to cope with everyday life. Further research on the topic is needed to meet the care needs of this patient group.