published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

Liver transplantation is the only life-saving treatment for patients with end-stage liver disease; however, organ availability is insufficient to meet demands. Steatotic livers are extended criteria donor (ECD) organs that could be used for transplantation if not for an increased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene, that when upregulated has be shown to reduce IRI in animal models of transplantation. Increasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating HO-1 protein (through the use of cell-penetrating peptides) may provide protection against IRI, making these organs useful for transplantation. The purpose of this thesis was the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene sequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT sequence. Resulting plasmids were cloned into E. coli, and protein expression was induced using IPTG. Proteins were purified using Ni-NTA affinity chromatography under denaturing and non-denaturing conditions. Non-denatured proteins were tested for HO-1 activity and the ability of both denatured and non-denatured proteins to transduce cells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured proteins was further tested in J774, HepG2 and HUVEC cells using immunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated expressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic activity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than their denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate multiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1 protein, for use in an animal model of steatotic liver transplantation. This protein demonstrates promise for use as a potential therapeutic agent in the field of liver transplantation.

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It is important to account for all sources of uncertainty when evaluating the clinical or cost-effectiveness of health care technologies. Therefore, this thesis takes as its basis a cost-effectiveness study in liver transplantation and identifies two previously unexplored issues that can arise in clinical and cost-effectiveness studies. A literature review of studies evaluating the effectiveness, costs or cost-effectiveness of solid organ transplantation confirmed that these issues were important and relevant to other transplantation studies. The first issue concerns the selection of an appropriate method for estimating mean study costs in the presence of incomplete (censored) data. Twelve techniques were identified and their accuracy was compared across artificially created mechanisms and levels of censoring. Lin's method with known cost histories and short interval lengths is recommended for accurately estimating mean costs and their uncertainty. It is assumed that these findings are generalisable to any solid organ transplant study where censoring is an issue. The second issue explored in this thesis relates to methods for measuring uncertainty around survival, HRQL and cost estimates derived from prognostic models in the absence of observed data. Probabilistic sensitivity analysis is recommended for measuring prognostic model parameter uncertainty and estimating individual patient outcomes and their uncertainties, as it is able to incorporate the additional uncertainty from using prognostic models to estimate control group outcomes. This thesis shows the quantitative importance of these issues and the methodological guidance offered should enable decision makers to have more confidence in clinical and cost-effectiveness estimates. Providing decision makers with a fuller estimate of the uncertainty around clinical and cost effectiveness estimates will aid them in decisions about the necessity of conducting further research in to the clinical or cost-effectiveness of health care technologies.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universtiet, 2010. / Härtill 4 uppsatser.

The diagnosis of antibody-mediated rejection after liver transplantation is complicated, due to the fact that the clinical and pathological signs of this life- threatening complication are often overlapping with non-immunological symptoms, like biliary obstruction, ischemia, thrombosis and others. Furthermore, the transplanted liver is to a great extent resistant to this type of rejection. Like in the transplanted kidney and heart, the main pathological factors of graft injury are antibodies directed to the mismatched HLA antigens of the organ donor, i.e. donor- specific antibodies. Besides analysis of HLA specificity of antibodies, research lately has been directed to define whether these antibodies are complement-binding or not. Literature data on this are however up till now limited. Therefore, the aim of this diploma thesis was to study the clinical relevance of complement-binding antibodies against HLA antigens in patients after liver transplantation. Our preliminary results suggest that there might be a correlation between the presence of complement-binding antibodies and the development of antibody-mediated rejection. This finding may play a role for improvement of the prognosis of patients after liver transplantation. Key words HLA, antibodies, C4d, complement, liver transplantation

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A regiÃo AmazÃnica à uma das principais Ãreas endÃmicas da hepatite D no Mundo e a Ãnica relacionada com a presenÃa do genÃtipo 3 do vÃrus delta. O objetivo deste estudo foi analisar os resultados do transplante de fÃgado pela cirrose causada pela hepatite D e correlacionar com os transplantados pela monoinfecÃÃo do vÃrus da hepatite B. Foram avaliados 29 pacientes com hepatite D e 40 pacientes com hepatite B, transplantados de fÃgado no Hospital UniversitÃrio Walter CantÃdio da Universidade Federal do Cearà (HUWC/UFC). O grupo de pacientes com hepatite D foi mais jovem (mÃdia 33.9 vs 52.9 anos, p < 0,001), predominante do sexo masculino e todos oriundos da RegiÃo AmazÃnica Brasileira. NÃo houve diferenÃas nos valores dos critÃrios de gravidade, MELD e Child, entre os grupos. A ocorrÃncia de carcinoma hepatocelular (CHC) foi predominante no grupo de pacientes com hepatite B ( frequÃncia 36.8% vs 17.2%, p = 0,05), assim como maior mortalidade ( frequÃncia 25% vs 3.4%, p = 0,019 ). Pacientes com hepatite D, apresentaram mais acentuada plaquetopenia no prÃ-transplante (mÃdia 66.428,57 vs 102.037,50 ÂL, p = 0,02) e no pÃs-operatÃrio imediato (mÃdia 54.242,86 vs 94.063,89 ÂL, p = 0,04). A recuperaÃÃo da lesÃo hepÃtica, avaliada atravÃs dos valores de AST, ALT e BT, mensurados 3 meses apÃs o transplante, nÃo mostrou diferenÃa entre os grupos. A sobrevida analisada pela curva de Kaplan-Meier, no perÃodo de 4 anos, foi de 95% nos pacientes com hepatite D e 75% nos pacientes com a monoinfecÃÃo pelo vÃrus B. Conclui-se que os pacientes com hepatite D possuem excelentes resultados apÃs o transplante hepÃtico e menor incidÃncia para o desenvolvimento do CHC. / The Amazon region is one of the main endemic areas of hepatitis D in the World and the only related to the presence of genotype 3 of delta virus. The objective of this study was to analyze the results of liver transplantation for cirrhosis caused by hepatitis D and correlate with the transplant by monoinfection of hepatitis B. 29 patients were evaluated with hepatitis D and 40 patients with hepatitis B, they were submitted a liver transplantation in the Walter Cantidio University Hospital of the Federal University of Ceara. The group of patients with hepatitis D were younger (mean 33.9 vs 52.9 years, p < 0,001), predominantly male and all from the Brazilian Amazon region. There were no differences in the values of severity criteria, MELD and Child, between groups. The occurrence of hepatocellular carcinoma (HCC) was predominant in patients with hepatitis B (frequency 36.8% vs. 17.2%, p = 0,05), likewise higher mortality (frequency 25% vs 3.4%, p = 0,019). Patients with hepatitis D showed more pronounced thrombocytopenia in pre-transplant (mean 66.428,57 vs 102.037,50 ÂL, p = 0,02) and in the immediate postoperative period (mean 54.242,86 vs 94.063,89 ÂL, p = 0,04). The recovery of liver injury as measured by the values of AST, ALT, and BT, measured 3 months after transplantation showed no difference between groups. The survival analyzed by Kaplan-Meier survival curves, showed a period of four years, the result of 95% in patients with hepatitis D and 75% in patients with monoinfection of hepatitis B. Patients with hepatitis D have excellent results after liver transplantation and lower incidence of HCC development.