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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18871号 / 医博第3982号 / 新制||医||1008(附属図書館) / 31822 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 三森 経世, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A partir da década de 60, com a utilização do transplante renal em larga escala como terapia substitutiva para pacientes com falência do órgão, surgiu a preocupação quanto ao desenvolvimento do processo de rejeição do enxerto. Tal intercorrência, em geral, cursa com sinais e sintomas clínicos apenas quando o evento está bem estabelecido, ou mesmo quando lesões irreversíveis já se instalaram. Assim, é fundamental um acompanhamento rigoroso, visando detectar os casos subclínicos. O presente trabalho, a fim de fornecer novas ferramentas que auxiliem o diagnóstico precoce de rejeição do enxerto, avaliou a expressão imuno-histoquímica dos anticorpos CD3, CD5, CD20, CD68, CD25, FoxP3 e C4d em biópsias renais realizadas entre os anos de 2007 e 2009 em pacientes transplantados acompanhados pelo Serviço de Nefrologia do Hospital Universitário Pedro Ernesto, UERJ - RJ, correlacionando os resultados obtidos com o diagnóstico histológico. Para tal, as biópsias foram reavaliadas por três médicos patologistas que as classificaram, segundo Critérios de Banff 2007, quanto à presença ou não de rejeição do enxerto e seu tipo, aguda ou crônica. A partir de então, os blocos de parafina foram processados pela técnica Tissue Microarray for all (Pires, ARC. e cols.) e submetidos à imuno-histoquímica. A positividade dos marcadores foi avaliada e graduada e os resultados encontrados foram correlacionados, em um primeiro momento, com a presença ou ausência de rejeição. Posteriormente, os casos com diagnóstico histológico de rejeição tiveram seu perfil imuno-histoquímico analisado em função da positividade para C4d, marcador definidor de rejeição humoral. Neste momento, buscou-se averiguar se os anticorpos estudados seriam úteis em detectar, neste grupo, rejeição humoral e celular. Após a análise estatística, realizada pelo Teste Exato de Fisher, pode-se, então, concluir que o comportamento do marcador CD3 é capaz de inferir a presença de rejeição e que os anticorpos CD5 e CD25 permitem sugerir rejeição celular e humoral, respectivamente. Foi observado também que casos sem diagnóstico histológico de rejeição podem apresentar marcação para C4d em mais de 10% de seus capilares peritubulares. / From the 60's, with the use of renal transplantation on a large scale as replacement therapy for patients with organ failure, came out the concern about the development process of graft rejection. This intercurrence, generally, evolves with clinical signs and symptoms only when the event is well established, or even when irreversible damage has already been installed. So, is essential a close monitoring, looking forward to detect subclinical cases. The present work, in order to provide new tools that help in the early diagnosis of rejection of the graft, evaluated the immunohistochemical expression of CD3, CD5, CD20, CD68, CD25, FoxP3 and C4d in renal biopsies performed between the years 2007 and 2009 in transplanted patients accompanied by the Department of Nephrology of Pedro Ernesto University Hospital, UERJ - RJ, correlating the results with the histological diagnosis. To this, the biopsies were evaluated by three pathologists who classified them, according to Banff 2007 Criteria, for the presence or absence of graft rejection and its type, acute or chronic. Thereafter, the paraffin blocks were processed by Tissue Microarray for all technique (Pires, ARC. & cols.) and submitted to immunohistochemistry. The positivity of the markers was evaluated and graded and the results were correlated, at first, with the presence or absence of rejection. Later, cases with histological diagnosis of rejection had their immunohistochemical profile considered according to the positivity for C4d, a defining marker of humoral rejection. At this point, we sought to determine whether the antibodies would be useful in detecting, in this studied group, humoral and cellular rejection. After statistical analysis, performed by Fisher's Exact Test, it could be, therefore, concluded that the behavior of the CD3 marker is able to infer the presence of rejection and that CD5 and CD25 antibodies may suggest cellular and humoral rejection, respectively. It was also observed that cases without histological diagnosis of rejection may have markings for C4d in more than 10% of their peritubular capillaries.

A partir da década de 60, com a utilização do transplante renal em larga escala como terapia substitutiva para pacientes com falência do órgão, surgiu a preocupação quanto ao desenvolvimento do processo de rejeição do enxerto. Tal intercorrência, em geral, cursa com sinais e sintomas clínicos apenas quando o evento está bem estabelecido, ou mesmo quando lesões irreversíveis já se instalaram. Assim, é fundamental um acompanhamento rigoroso, visando detectar os casos subclínicos. O presente trabalho, a fim de fornecer novas ferramentas que auxiliem o diagnóstico precoce de rejeição do enxerto, avaliou a expressão imuno-histoquímica dos anticorpos CD3, CD5, CD20, CD68, CD25, FoxP3 e C4d em biópsias renais realizadas entre os anos de 2007 e 2009 em pacientes transplantados acompanhados pelo Serviço de Nefrologia do Hospital Universitário Pedro Ernesto, UERJ - RJ, correlacionando os resultados obtidos com o diagnóstico histológico. Para tal, as biópsias foram reavaliadas por três médicos patologistas que as classificaram, segundo Critérios de Banff 2007, quanto à presença ou não de rejeição do enxerto e seu tipo, aguda ou crônica. A partir de então, os blocos de parafina foram processados pela técnica Tissue Microarray for all (Pires, ARC. e cols.) e submetidos à imuno-histoquímica. A positividade dos marcadores foi avaliada e graduada e os resultados encontrados foram correlacionados, em um primeiro momento, com a presença ou ausência de rejeição. Posteriormente, os casos com diagnóstico histológico de rejeição tiveram seu perfil imuno-histoquímico analisado em função da positividade para C4d, marcador definidor de rejeição humoral. Neste momento, buscou-se averiguar se os anticorpos estudados seriam úteis em detectar, neste grupo, rejeição humoral e celular. Após a análise estatística, realizada pelo Teste Exato de Fisher, pode-se, então, concluir que o comportamento do marcador CD3 é capaz de inferir a presença de rejeição e que os anticorpos CD5 e CD25 permitem sugerir rejeição celular e humoral, respectivamente. Foi observado também que casos sem diagnóstico histológico de rejeição podem apresentar marcação para C4d em mais de 10% de seus capilares peritubulares. / From the 60's, with the use of renal transplantation on a large scale as replacement therapy for patients with organ failure, came out the concern about the development process of graft rejection. This intercurrence, generally, evolves with clinical signs and symptoms only when the event is well established, or even when irreversible damage has already been installed. So, is essential a close monitoring, looking forward to detect subclinical cases. The present work, in order to provide new tools that help in the early diagnosis of rejection of the graft, evaluated the immunohistochemical expression of CD3, CD5, CD20, CD68, CD25, FoxP3 and C4d in renal biopsies performed between the years 2007 and 2009 in transplanted patients accompanied by the Department of Nephrology of Pedro Ernesto University Hospital, UERJ - RJ, correlating the results with the histological diagnosis. To this, the biopsies were evaluated by three pathologists who classified them, according to Banff 2007 Criteria, for the presence or absence of graft rejection and its type, acute or chronic. Thereafter, the paraffin blocks were processed by Tissue Microarray for all technique (Pires, ARC. & cols.) and submitted to immunohistochemistry. The positivity of the markers was evaluated and graded and the results were correlated, at first, with the presence or absence of rejection. Later, cases with histological diagnosis of rejection had their immunohistochemical profile considered according to the positivity for C4d, a defining marker of humoral rejection. At this point, we sought to determine whether the antibodies would be useful in detecting, in this studied group, humoral and cellular rejection. After statistical analysis, performed by Fisher's Exact Test, it could be, therefore, concluded that the behavior of the CD3 marker is able to infer the presence of rejection and that CD5 and CD25 antibodies may suggest cellular and humoral rejection, respectively. It was also observed that cases without histological diagnosis of rejection may have markings for C4d in more than 10% of their peritubular capillaries.

La greffe cardiaque est le traitement ultime de l’insuffisance cardiaque. Le rejet aigu pose plusieurs problématiques, en particulier sa survenue imprévisible même sous traitement immunosuppresseur, et un diagnostic histologique qui nécessite des biopsies endomyocardiques (BEM) invasives répétées, et qui souffre de nombreuses limites. Le besoin de critères diagnostiques et prédictifs, idéalement non invasifs, nous a conduits à étudier le rejet aigu de greffe cardiaque sur le plan moléculaire. Nous avons caractérisé les profils d’expression génique (PEG) myocardiques et sanguins lors de différentes phases du rejet cellulaire (RC) et du rejet médié par les anticorps (RMA), par analyse sans a priori des transcriptomes sur puce à ADN. Par une première étude des PEG myocardiques menée sur une collection historique de BEM, nous avons montré la modification des PEG tissulaires lors du RC. Pour le même grade histologique, deux profils de RC aux degrés d’activation immunitaire différents ont été identifiés. De plus, les PEG myocardiques étaient modifiés dès un mois avant la survenue d’un RC, quand l’analyse histologique ne montrait encore aucune anomalie. Par une seconde étude conduite sur une collection prospective de BEM et échantillons sanguins, nous avons confirmé les résultats de la première étude, et de plus montré l’existence de modulations des PEG également dans le sang périphérique, aussi bien pendant un épisode de RC qu’un mois avant. Enfin pour la première fois la modulation tissulaire et périphérique des PEG a été montrée dans le RMA en transplantation cardiaque. L’existence de voies modulées dans les deux types de rejet devrait conduire à la recherche de biomarqueurs. / Heart transplantation is the last treatment in case of terminal heart failure. Acute rejection after heart transplantation raises several issues due to its occurrence despite immunosuppressive therapies and the requirement of invasive and repeated endomyocardial biopsies (EMB) that have several histological grading limitations. The need of non-invasive diagnostic and predictive criteria led us to study the acute rejection of cardiac allograft using a molecular approach. We characterized myocardial and peripheral blood gene expression profiles (GEP) during acute cellular rejection (CR) and antibody-mediated rejection (AMR) by mean of microarray analyses. By a retrospective study conducted on a historical EMB collection, we first showed a strong immunologic modulation during CR. For the same CR histological grading, two transcriptional profiles were identified according to the inflammation level. Moreover, myocardial GEP modifications were observed one month before the occurrence of CR, while histological characteristics showed no abnormality. A second study conducted on a prospective collection of both EMB and peripheral blood samples confirmed the results obtained on EMB and showed peripheral blood GEP modulations during both CR and even one month earlier. Finally, we have also shown for the first time in heart transplantation, myocardial and peripheral GEP modulations in AMR. Identification of modulated pathways in both types of rejection should allow for the determination of rejection biomarkers.

Since its beginning, graft rejection remains the key problem of solid organ transplantation. This reaction of the recipient's immune system against mismatched antigens of the transplanted organ causes graft damage and consequently loss of its function. Rejection involves cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Among the genetic factors which may have a prognostic value in rejection risk evaluation are the Human Leukocyte Antigens (HLA) genotype, the Killer Immunoglobuline-like Receptor (KIR) gene repertoir, cytokine and other gene polymorphisms. These factors could be screened for before transplantation to find the best possible combination of genetic characteristics of the donor and recipient and to reveal patients with "risky" genotypes, who may need more intensive immunosuppression and more careful post-transplant follow-up. Molecular factors, such as HLA and non-HLA antibodies, soluble CD30 molecule (sCD30), Hepatocyte Growth Factor (HGF) and other cytokines, measured before and/or after transplantation in the recipient's blood may be helpful for rejection risk estimation and may also be used as post-transplant rejection onset markers. In our study, we focused on some of the above mentioned factors. We found that ethnicity plays a significant role in the...