Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

Long-term survival of cardiac transplant recipients continues to be severely limited by the development of a pathological, chronic rejection process, termed allograft vasculopathy (AV). This remains to be the case despite dramatic improvements in the areas of surgical techniques, pre- and post-operative care, and immunosuppression. To model the clinical setting we used calcineurin inhibitor (CNI) immunosuppression, the cornerstone of post-transplant immunosuppression, in a murine aortic interposition transplant model for our analysis of AV development. This model mimics the presentation of AV in human cardiac transplants through the development of a progressively occlusive neointimal lesion. Our previous work in this model has demonstrated that CD8+, but not CD4+, T cells play a role in neointimal lesion formation. Further investigation also highlighted a specific requirement for either CD8+ T cell-derived IFN-γ or direct cytotoxicity in the development of lesion formation. In the current study we confirmed that CD8+ T cell-derived IFN-γ also leads to the loss of medial smooth muscle cells, an event which inversely correlates with lesion formation. The Fas/FasL direct cytotoxic pathway was also significantly involved in neointimal lesion formation and medial remodeling. This work clarified the pathways utilized by CD8+ T cells in their role as mediators of AV development. Recognizing the threat that CD8+ T cells pose to cardiac transplant recipients in the presence of CNI immunosuppression, and a growing concern with the presence of anti-donor memory T cells in transplant recipients, we next explored the development of memory CD8+ T cells in the presence of CNI immunosuppression. We first established that memory CD8+ T cells could not develop when CNI immunosuppression was initiated immediately post-challenge. Next, we hypothesized that the clinical practice of CNI delay post-transplant would permit the development of de novo memory CD8+ T cells. Immediate and early initiation was sufficient to prevent the development of de novo memory CD8+ T cells. However, later delay to within a clinically practiced timeframe did permit the development of de novo memory CD8+ T cells. Our analysis revealed that this population demonstrated equivalent functionality to de novo memory CD8+ T cells generated in the absence of CNI immunosuppression.

Proposição: avaliar radiograficamente e histologicamente o comportamento do incisivo inferior de rato frente à realização de uma solução de continuidade em sua porção radicular mediana quanto ao comportamento do tecido pulpar e dos enxertos ósseos alógenos congelados à -80°C e a evolução do seu processo eruptivo dentário em alvéolos quando expostos aos efeitos da ação do ultra-som. Metodologia: estudo experimental in vivo, amostragem randomizada, com um grupo teste e outro controle. Constou de 29 ratos, da espécie Rattus novergicus albinus, linhagem Wistar, machos, divididos em quatro grupos, correspondendo aos tempos de sete, 14, 21 e 45 dias. Em cada grupo, cinco animais foram teste e dois animais foram controle. Em cada animal foi removido, cirurgicamente, o segmento medial do incisivo inferior direito. Entretanto, nos animais do grupo experimental foram realizados enxertos de osso alógeno congelado a -80°C, nos 2,0mm distais do total da cavidade alveolar cirurgicamente obtida com ultra-som.Resultados: aos sete dias observou-se estrutura tecidual desorganizada e ricamente vascularizada em ambos os grupos avaliados e início da integração do enxerto ósseo no grupo teste. Aos 14 dias seguem as mesmas observações descritas aos sete dias com progressão da incorporação do enxerto ósseo. Aos 21 dias, observa-se intensa neoformação óssea, ricamente vascularizada em ambos grupos. Aos 45 dias, existe também uma intensa neoformação óssea alveolar, fechando o alvéolo e a completa integração dos enxertos ósseos. Em todos os grupos observou-se que houve mínimo crescimento do segmento dentário proximal e que o mesmo manteve sua vitalidade pulpar. Conclusão: Após a realização deste estudo foi possível concluir que: o tecido pulpar, contido no segmento proximal manteve sua vitalidade, reagindo na forma de reparação dentinária compatível com o dente humano; o enxerto ósseo alógeno congelado evolui favoravelmente no processo de incorporação a partir de um leito receptor criado no alvéolo dentário e que o incisivo inferior do rato, submetido à odontossecção com ultra-som não seguiu com o processo de crescimento e erupção em seu segmento proximal, podendo estar relacionado com o reparo do alvéolo devido à intensa neoformação óssea, provavelmente por efeito do ultra-som. / Proposition: This study aimed to evaluate, both radiographically and histologically, the behaviour of a rat’s inferior incisive concerning the realization of a continuous solution in its medial radicular portion, regarding the behaviour of the pulpal tissue and allogeneic freezing bone grafts, at –80° C, as well as the evolution in its dental eruptive process in alveoli exposed to ultrasound effects. Methodology: An in vivo experimental study, using randomized samples, including a study group and a control group. Twenty-nine Wistar strain male rats, Rattus novergicus albinus species, divided into 4 groups, corresponding to 7, 14, 21, and 45 days. From each group, five animals were considered as the test group, and two of them as the control group. Each animal had the medial segment of the right inferior incisive surgically removed. However, the animals from the test group underwent allogeneic freezing bone grafts, at –80° C, in a range of 2.0 mm distal of the total alveolar cavity, surgically performed by ultrasound. Results: In seven days a disorganized and well-vascularized tissue structure in both assessment groups as well as the beginning of the integration of the bone graft in the experimental group could be observed. In 14 days similar observations to day 7 and progression in the incorporation of the bone graft were found. In 21 days intense, well-vascularized, bone neoformation was observed in both groups. In 45 days an intense neoformation of the alveolar bone could also be observed, with the closure of the alveolus, and the complete integration of the bone grafts. All groups had a minimum growth of the dental proximal segment, which maintained its pulpal vitality. Conclusion: The study concluded that the pulpal tissue contained in the proximal segment maintained its vitality, providing a reaction compatible with human tooth regarding dental repairing. The allogeneic freezing bone graft evolved favorably in the incorporation process from a receiver layer created inside the dental alveolus. It was also concluded the rat’s inferior incisive which underwent ultrasonic dental cutting did not follow the growth and eruption process in its proximal segment, what might be related to the alveolus repair due to intense bone neoformation, probably caused by the ultrasound effect.

Proposição: avaliar radiograficamente e histologicamente o comportamento do incisivo inferior de rato frente à realização de uma solução de continuidade em sua porção radicular mediana quanto ao comportamento do tecido pulpar e dos enxertos ósseos alógenos congelados à -80°C e a evolução do seu processo eruptivo dentário em alvéolos quando expostos aos efeitos da ação do ultra-som. Metodologia: estudo experimental in vivo, amostragem randomizada, com um grupo teste e outro controle. Constou de 29 ratos, da espécie Rattus novergicus albinus, linhagem Wistar, machos, divididos em quatro grupos, correspondendo aos tempos de sete, 14, 21 e 45 dias. Em cada grupo, cinco animais foram teste e dois animais foram controle. Em cada animal foi removido, cirurgicamente, o segmento medial do incisivo inferior direito. Entretanto, nos animais do grupo experimental foram realizados enxertos de osso alógeno congelado a -80°C, nos 2,0mm distais do total da cavidade alveolar cirurgicamente obtida com ultra-som.Resultados: aos sete dias observou-se estrutura tecidual desorganizada e ricamente vascularizada em ambos os grupos avaliados e início da integração do enxerto ósseo no grupo teste. Aos 14 dias seguem as mesmas observações descritas aos sete dias com progressão da incorporação do enxerto ósseo. Aos 21 dias, observa-se intensa neoformação óssea, ricamente vascularizada em ambos grupos. Aos 45 dias, existe também uma intensa neoformação óssea alveolar, fechando o alvéolo e a completa integração dos enxertos ósseos. Em todos os grupos observou-se que houve mínimo crescimento do segmento dentário proximal e que o mesmo manteve sua vitalidade pulpar. Conclusão: Após a realização deste estudo foi possível concluir que: o tecido pulpar, contido no segmento proximal manteve sua vitalidade, reagindo na forma de reparação dentinária compatível com o dente humano; o enxerto ósseo alógeno congelado evolui favoravelmente no processo de incorporação a partir de um leito receptor criado no alvéolo dentário e que o incisivo inferior do rato, submetido à odontossecção com ultra-som não seguiu com o processo de crescimento e erupção em seu segmento proximal, podendo estar relacionado com o reparo do alvéolo devido à intensa neoformação óssea, provavelmente por efeito do ultra-som. / Proposition: This study aimed to evaluate, both radiographically and histologically, the behaviour of a rat’s inferior incisive concerning the realization of a continuous solution in its medial radicular portion, regarding the behaviour of the pulpal tissue and allogeneic freezing bone grafts, at –80° C, as well as the evolution in its dental eruptive process in alveoli exposed to ultrasound effects. Methodology: An in vivo experimental study, using randomized samples, including a study group and a control group. Twenty-nine Wistar strain male rats, Rattus novergicus albinus species, divided into 4 groups, corresponding to 7, 14, 21, and 45 days. From each group, five animals were considered as the test group, and two of them as the control group. Each animal had the medial segment of the right inferior incisive surgically removed. However, the animals from the test group underwent allogeneic freezing bone grafts, at –80° C, in a range of 2.0 mm distal of the total alveolar cavity, surgically performed by ultrasound. Results: In seven days a disorganized and well-vascularized tissue structure in both assessment groups as well as the beginning of the integration of the bone graft in the experimental group could be observed. In 14 days similar observations to day 7 and progression in the incorporation of the bone graft were found. In 21 days intense, well-vascularized, bone neoformation was observed in both groups. In 45 days an intense neoformation of the alveolar bone could also be observed, with the closure of the alveolus, and the complete integration of the bone grafts. All groups had a minimum growth of the dental proximal segment, which maintained its pulpal vitality. Conclusion: The study concluded that the pulpal tissue contained in the proximal segment maintained its vitality, providing a reaction compatible with human tooth regarding dental repairing. The allogeneic freezing bone graft evolved favorably in the incorporation process from a receiver layer created inside the dental alveolus. It was also concluded the rat’s inferior incisive which underwent ultrasonic dental cutting did not follow the growth and eruption process in its proximal segment, what might be related to the alveolus repair due to intense bone neoformation, probably caused by the ultrasound effect.

Proposição: avaliar radiograficamente e histologicamente o comportamento do incisivo inferior de rato frente à realização de uma solução de continuidade em sua porção radicular mediana quanto ao comportamento do tecido pulpar e dos enxertos ósseos alógenos congelados à -80°C e a evolução do seu processo eruptivo dentário em alvéolos quando expostos aos efeitos da ação do ultra-som. Metodologia: estudo experimental in vivo, amostragem randomizada, com um grupo teste e outro controle. Constou de 29 ratos, da espécie Rattus novergicus albinus, linhagem Wistar, machos, divididos em quatro grupos, correspondendo aos tempos de sete, 14, 21 e 45 dias. Em cada grupo, cinco animais foram teste e dois animais foram controle. Em cada animal foi removido, cirurgicamente, o segmento medial do incisivo inferior direito. Entretanto, nos animais do grupo experimental foram realizados enxertos de osso alógeno congelado a -80°C, nos 2,0mm distais do total da cavidade alveolar cirurgicamente obtida com ultra-som.Resultados: aos sete dias observou-se estrutura tecidual desorganizada e ricamente vascularizada em ambos os grupos avaliados e início da integração do enxerto ósseo no grupo teste. Aos 14 dias seguem as mesmas observações descritas aos sete dias com progressão da incorporação do enxerto ósseo. Aos 21 dias, observa-se intensa neoformação óssea, ricamente vascularizada em ambos grupos. Aos 45 dias, existe também uma intensa neoformação óssea alveolar, fechando o alvéolo e a completa integração dos enxertos ósseos. Em todos os grupos observou-se que houve mínimo crescimento do segmento dentário proximal e que o mesmo manteve sua vitalidade pulpar. Conclusão: Após a realização deste estudo foi possível concluir que: o tecido pulpar, contido no segmento proximal manteve sua vitalidade, reagindo na forma de reparação dentinária compatível com o dente humano; o enxerto ósseo alógeno congelado evolui favoravelmente no processo de incorporação a partir de um leito receptor criado no alvéolo dentário e que o incisivo inferior do rato, submetido à odontossecção com ultra-som não seguiu com o processo de crescimento e erupção em seu segmento proximal, podendo estar relacionado com o reparo do alvéolo devido à intensa neoformação óssea, provavelmente por efeito do ultra-som. / Proposition: This study aimed to evaluate, both radiographically and histologically, the behaviour of a rat’s inferior incisive concerning the realization of a continuous solution in its medial radicular portion, regarding the behaviour of the pulpal tissue and allogeneic freezing bone grafts, at –80° C, as well as the evolution in its dental eruptive process in alveoli exposed to ultrasound effects. Methodology: An in vivo experimental study, using randomized samples, including a study group and a control group. Twenty-nine Wistar strain male rats, Rattus novergicus albinus species, divided into 4 groups, corresponding to 7, 14, 21, and 45 days. From each group, five animals were considered as the test group, and two of them as the control group. Each animal had the medial segment of the right inferior incisive surgically removed. However, the animals from the test group underwent allogeneic freezing bone grafts, at –80° C, in a range of 2.0 mm distal of the total alveolar cavity, surgically performed by ultrasound. Results: In seven days a disorganized and well-vascularized tissue structure in both assessment groups as well as the beginning of the integration of the bone graft in the experimental group could be observed. In 14 days similar observations to day 7 and progression in the incorporation of the bone graft were found. In 21 days intense, well-vascularized, bone neoformation was observed in both groups. In 45 days an intense neoformation of the alveolar bone could also be observed, with the closure of the alveolus, and the complete integration of the bone grafts. All groups had a minimum growth of the dental proximal segment, which maintained its pulpal vitality. Conclusion: The study concluded that the pulpal tissue contained in the proximal segment maintained its vitality, providing a reaction compatible with human tooth regarding dental repairing. The allogeneic freezing bone graft evolved favorably in the incorporation process from a receiver layer created inside the dental alveolus. It was also concluded the rat’s inferior incisive which underwent ultrasonic dental cutting did not follow the growth and eruption process in its proximal segment, what might be related to the alveolus repair due to intense bone neoformation, probably caused by the ultrasound effect.

Solid organ transplantation is now an established and effective treatment option for end-stage organ failure. Whilst early outcomes have improved significantly over recent decades, longer-term outcomes have changed little. Despite advances in immunosuppression, most transplanted organs suffer an inevitable decline in function attributed to chronic rejection. It is evident that the alloimmune response remains incompletely characterised. Crucially, despite description several decades ago, the precise contribution that the direct (recognition of intact allogeneic MHC) and indirect (recognition of self-MHC restricted allopeptide) pathways make to allograft rejection remains incompletely understood. In this thesis, murine models of heterotopic cardiac transplantation have been utilised to analyse these pathways. The key findings of this work are as follows: 1) If able to evade NK cell killing, passenger donor CD4 T cells can make cognate, direct-pathway, interactions with recipient B cells. This interaction results in augmentation of all arms of the alloimmune response and acceleration of allograft rejection. 2) Direct-pathway CD4 T cell allorecognition is restricted to the immediate post transplantation period. Donor APCs are the major source of MHC class II for direct-pathway priming, and these are cleared rapidly by both innate and adaptive responses of the recipient, effectively limiting the longevity of direct allorecognition. 3) The duration of indirect-pathway responses against different alloantigens is variable, limited by availability of donor antigen. Expression of donor MHC class II is restricted to APCs and possibly endothelium (where expression is transient) limiting the duration of indirect-pathway allorecognition against MHC class II alloantigen. Indirect-pathway CD4 T cell responses targeted against parenchymal alloantigen are long-lived, and can provide help for generating alloantibody against different MHC alloantigens. 4) In response to continual presentation of target epitope indirect-pathway CD4 T cell responses against parenchymal expressed alloantigen are long-lived. The continual division of these cells results in greatly increased numbers of alloantigen-specific CD4 T cells in the chronic phase of the response, but despite this, memory responses are impaired. 5) Generating indirect-pathway regulatory T cells specific for parenchymal expressed alloantigen appears to be the most effective strategy to ameliorating chronic rejection.

BACKGROUND. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor for coronary artery disease (CAD) in nontransplant patients. We evaluated the association between Lp-PLA2, cardiac allograft vasculopathy (CAV) assessed by 3D intravascular ultrasound, and incidence of cardiac adverse events in heart transplant recipients. MATERIALS AND METHODS. Fasting blood samples were obtained and stored from a cross-section of 112 cardiac transplant recipients attending the Mayo cardiac transplant clinic in 2000 to 2001, mean of 4.7 years after transplant. Lp-PLA2 was measured in plasma aliquots using an enzyme-linked immunoassay. Fifty-six of these patients subsequently underwent two 3D intravascular ultrasound studies in 2004 to 2006 12 months apart. Cardiovascular (CV) events included percutaneous coronary intervention, coronary artery bypass grafting (CABG), reduction in left ventricular ejection fraction (LVEF) ≤45% secondary to CAV and CV death. RESULTS. High Lp-PLA2 level was associated with increase in plaque volume (r=0.43, P=0.0026) and percent plaque volume (r=0.45, P=0.0004). The association remained significant after adjusting for clinical and lipid variables. During follow-up of 5.1±1.6 years, 24 CV adverse events occurred in 15 of 112 (13%) heart transplant patients. Lp-PLA2 level>236 ng/mL (higher tertile) identified a subgroup of patients having a 2.4-fold increase of relative risk for combined endpoint of CV events (percutaneous coronary intervention, CABG, LVEF<45%, and CV death; 95% CI 1.16-5.19, P=0.012) compared with patients with Lp-PLA2≤236 ng/mL. CONCLUSIONS. Lp-PLA2 is independently associated with progression of CAV and predicts a higher incidence of CV events and CV death in transplant patients. This finding supports the concept that systemic inflammation is an important mediator of CAV. Lp-PLA2 may be a useful marker for risk of CAV and a therapeutic target in posttransplant patients.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12958号 / 論医博第2100号 / 新制||医||1011(附属図書館) / 32357 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三嶋 理晃, 教授 三森 経世, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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