1 resultado para Óxido nítrico sintase Teses
Resumo:
RESUMO: O transplante heptico ortotpico uma teraputica aceite para casos selecionados de falncia heptica terminal. O procedimento tem-se aperfeioado, evidenciado pelo aumento da taxa de sobrevida de 30 para 75% aos 5 anos, mas cerca de 13 a 27% dos enxertos desenvolve falncia primria (PNF) ou disfuno primria (DF) aps o transplante. As consequncias so devastadoras para a sobrevida do doente e do enxerto. A sua etiologia multifactorial, incluindo factores relacionados com o dador e o receptor, tempos de isqumia, agresses cirrgicas, bem como caractersticas anatomopatolgicas do enxerto. A leso de isqumia/reperfuso mantem-se como um factor de risco intra operatrio, com implicaes directas sobre toda a evoluo do transplante : existe uma relao ntima entre a PNF e a DF, a preservao do enxerto, a leso de isqumia/reperfuso, e a falncia do transplante. Alm disso, est comprovada evidncia que sugere que a leso de I/R torna um aloenxerto mais vulnervel por aumento da imunogenicidade, aumentando a probabilidade de episdios de rejeio precoce e tardia. Com base na prtica clnica quotidiana do CHBPT HCC, estudaram-se 54 casos de transplante heptico, agrupados segundo grupos por alocao do enxerto respectivo: Grupo 1(n=27): dador cadver para receptor cirrtico, Grupo 2 (n=15): dador cadver para receptor PAF, Grupo 3 (n=12): dador PAF para receptor cirrtico. Observaram-se as alteraes histolgicas e moleculares sobre o enxerto at ao final da operao do receptor, e as suas consequncias clnicas,avaliando: - As diferentes capacidades de resistncia e cada enxerto leso de isqumia/reperfuso. - As situaes em que os factores do receptor se sobrepem s do enxerto na definio do prognstico, e vice versa. - A relevncia das leses histolgicas e moleculares precoces no tecido heptico na evoluo do enxerto e do receptor. Foram colhidas bipsias por agulha dos 54 enxertos hepticos,42 provenientes de cadver com corao batente(morte cerebral) e 12 provenientes de dador vivo com PAF, em trs tempos diferentes do processo de colheita e transplante heptico: A primeira(T0)antes da clampagem da aorta do dador - A segunda (T1) no final da isqumia fria - A terceira (T2) aps a reperfuso do enxerto, durante o encerramento da parede abdominal. A estas amostras foi extrado RNA total, convertido em cDNA por transcrio reversa e feita a anlise da expresso dos genes da CTLA4, IL-1, IL-4, IL-6, IL-13, TNF-, Perforina, Selectina, (SELE), Fas-ligando, Granzima-B, Heme-Oxigenase 1(HO1)e Óxido Nítrico Sintetase(iNOS2A)por PCR quantitativo segundo o mtodo do Ct comparativo, utilizando como referncia a expresso dos genes da amostra no-isqumica T0. Os fragmentos de todas as bipsias foram seccionados, para envio de amostra comparativa para processamento histolgico habitual, sem qualquer alterao ao protocolo seguido habitualmente na Unidade de Transplantao do Hospital Curry Cabral. A presena de alguns parmetros histolgicos definidos, como esteatose, necrose, vacuolizao, congesto sinusoidal e infiltrao neutroflica, foi registada e contabilizada numa classificao numrica. O seguimento clnico e laboratorial, bem como o acompanhamento de eventuais complicaes, foi registado e correlacionado com os dados das colheitas de rgos e com os dados das bipsias. Foram consideradas as seguintes variveis, como as mais relevantes e objectivas para a interpretao da evoluo clnica, tendo sido comparadas estatisticamente com os dados recolhidos, laboratoriais e clnicos: disfuno do enxerto, 207 ps operatrias, nmero de internamentos igual ou superior a 2 e rejeio crnica e/ou morte do receptor. Foram identificadas caractersticas clnicas menos favorveis, a considerar, nalgumas circunstncias: gnero feminino do receptor (sobretudo associado a enxerto masculino, p=0,077), isqumia fria superior a 500 minutos (p=0,074), isqumia quente superior a 90 minutos (p=0,099). Na anlise laboratorial, distinguiram-se duas caractersticas histolgicas desfavorveis e irreversveis, como ndice de mau prognstico: a necrose e a balonizao (p=0,029); no painel gentico escolhido neste estudo,a expresso basal de IL-1(p=0,028), de SELE p=0,013)e de FAS-L (p=0,079)relacionaram-se com pior prognstico. Algumas caractersticas protectoras intrnsecas dos enxertos s se revelaram indirectamente, como menor infiltrao neutroflica e maior expresso de HO1 e de iNOS nos enxertos PAF, no tendo sido possvel provar uma interferncia directa nos resultados clnicos. No se obteve expresso mensurvel de genes anti- inflamatrios nas biopsias hepticas processadas neste estudo, como a IL13 e a I 4: assim, com a metodologia utilizada, no foi possvel obter um perfil de expresso gentica associado a boa evoluo clnica. O perfil inverso foi sugerido apenas pela expresso basal dos 3 genes mencionados (FAS-L,IL-1 e SELE)no mesmo painel, com o protocolo seguido neste conjunto de 54 doentes. As caractersticas do receptor sobrepuseram-se s do enxerto no caso de: - diagnstico de PAF no receptor, que determinou uma maior predisposio para a disfuno do enxerto, o que, por sua vez, determina uma menor sobrevida. No entanto, o diagnstico de PAF no receptor exibe uma curva de sobrevida mais favorvel. - receptores com um baixo balano de risco (BAR)definiram caractersticas favorveis para enxertos com nveis baixos e moderados de esteatose, fazendo que esta caracterstica, definida como um risco acrescido, no s no se manifestasse clinicamente,como parecesse um factor favorvel. As caractersticas do enxerto sobrepuseram-se s do receptor no caso de: - tempo de isqumia fria superior a 500 minutos - balonizao, necrose, FAS-L,IL-1 e SELE em T0 A integrao dos resultados moleculares e morfolgicos com a evoluo clnica, reala o papel da mobilizao precoce de neutrfilos nos desempenhos menos favorveis do enxerto heptico. -------------ABSTRACT: Orthotopic liver transplantation is na accepted therapeutic procedure for selected cases of terminal liver failure. The procedure has been improved, evidenced by the rise of survival rates from 30 to 70% at 5 years, but 13 to 27% of the liver grafts develops primary non function (PNF) or primary dysfunction (PDF) after transplantation. The consequences are devastating for the survival of the patient and of the graft. Its etiology is multifactorial, including factos related with the donor and with the recipient, ischemic times, surgical aggressions, as well as the histological characteristics of the graft. The ischemia/reperfusion lesion is still an intraoperative risk factor, with direct implications in the whole transplant outcome: there is a close interrelation between PNF and DF, graft preservation, ischemia / reperfusion lesion and graft failure. Beyond his, there is proved evidence that suggests that I/R lesion turns the allograft more vulnerable by increasing its immunogenity, increasing the probability of precocious and late rejection episodes. Based on the daily clinical practice at CHBPT /HCC, 54 cases of hepatic transplantation have been studied, grouped by allocation of each graft: Group (n=27):deceased do nortocirrhotic recipient, Group 2 (n=15): deceased donor to FAP recipient, Group 3 (n=12): FAP living donor to cirrhotic recipient. The histologic and molecular changes in the liver graft were observed until the end of the recipiente operation,together with its clinical consequences, evaluating:-The different capacity of resistance of each graft to the ischemia / reperfusion lesion - The situations where the recipiente factos overlap the ones of the graft, in the definition of prognosis, and vice versa.- The relevance of the precocious histologic and molecular lesions of the hepatic tissue in the clinical outcome of the graft and the recipient. Needle biopsies were obtained from 54 liver grafts, 42 deceased brain dead donors and 12 from FAP living donors, at three diferente times of the harvesting and the hepatic transplantation: The first one (T0) before clamping the donor aorta - The second one (T2) in the end of cold ischemia time - The third one (T) after the reperfusion of the graft, during the closure of the abdominal wall. Total RNAwas extracted to these samples, converted to cDNA by reverse transcription and the analysis of gene expression was made for CTLA4,IL-1,IL-4,IL-6,IL-13,TNF-,Perforin,E Selectin (SELE),Fas-ligand,Granzyme-B,Heme-oxigenase 1 (HO1) and Nitric Oxide Sintetase (iNOS2A) by quantitative PCR, according with the Ct comparative method, using the expression of the non ischemic sample T0. The fragments of all the biopsies were divided, to send a comparative sample to the usual histologic processement, keeping the same usual protocol at the Transplantation Unit of Curry Cabral Hospital. The presence of some defined histologic parameters, such as steatosis, necrosis, vacuolization, sinusoidal congestion and neutrophilic infiltration, was registered and catalogued in a numeric classification. The clinical and laboratory follow-up, as well as the following of eventual complications, was registered and correlated with the data from organ procurement operations and with the data from the biopsies. The following variables were considered as the most relevant and objective ones, to the interpretation of the clinical evolution, being statistically compared with the clinical and laboratorial collected data: graft dysfunction, post-operative complications, number of readmissions of 2 or more and chronic rejection and /or recipiente death. There were identified some unfavorable clinical characteristics, to be considered under certain circumstances: recipiente female gender (specially associated with malegraft, p=0,077), cold ischemia time of more than 500 minutes (p=0,074), warm ischemia time of more than 90 minutes (p=0,099). In the laboratory analysis, two histologic characteristics were identified as unfavorable and irreversible, associated with bad prognosis: necrosis and balonization (p=0,029); in the gene panel selected in this study, the basal expression of IL-1 (p=0,028), SELE (p=0,013) and FAS-L (p=0,079)were related with worse prognosis.Some intrinsic protective characteristics of the grafts were only indirectly revealed, such as less neutrophilic infiltration and bigger expression of HO1 and iNOS in FAP grafts, being impossible to prove any direct inte ference in the clinical results. A relevant and measurable expression of the anti inflammatory genes IL13 and IL4 was not obtained: with the used methodology, it was impossible to obtain a gene expression profile associated with a favorable clinical outcome.The inverse profile was suggested only by the basal expression of the three mentioned genes (FAS-L, IL- 1 e SELE) in the same gene panel, according with the followed protocol in this group of 54 patients. The characteristics of the recipient overlapped those from the graft, in the case of :- FAP diagnosis in the recipient, which determined a bigger predisposition to graft dysfunction, which by itself determines a shorter survival. However, FAP diagnosis in the recipiente depicts a more favorable survival curve. - Recipients with a low balance risk ndex (BAR) defined favorable characteristics to grafts with low and moderate grades of steatosis, making that this characteristic, associated with bad prognosis, looked like a favorable factor, and with no clinical interference. The graft characteristics overlapped those from the receptor in the case of: - Cold ischemic time more than 500 minutes - Balonization, necrosis, FAS-L, IL-1 and SELE at T0. The integration of molecular and morphologic results with the clinical evolution, stresses the role of a precocious neutrophils mobilization in the worse outcomes of liver grafts.
