INTRODUÇÃO: O transplante hepático (TxH) vem mudando o curso de doenças graves, incapacitantes e potencialmente fatais, se tornando o tratamento de escolha quando há falência do órgão. OBJETIVO: Comparar as taxas de TxH, exclusão e sobrevida entre candidatos com cirrose descompensada (CIR), situações especiais (SPE) e carcinoma hepatocelular (HCC). MÉTODOS:Foram realizados dois estudos em 358 pacientes: uma coorte retrospectiva (agosto de 2008 - julho de 2009, incluindo 189 pacientes listados) e uma prospectiva (de novembro de 2012 a maio de 2014, com um período de acompanhamento até novembro de 2015, incluindo 169 candidatos a transplante hepático, comparando CIR, HCC e SPE. Foram avaliadas as seguintes variáveis: K-in (taxas de entrada da lista de espera: K-1in se CIR, K-2in se HCC e K-3in se SPE); K-out (taxa de TxH); K-1out (drop out no grupo CIR); K-2out (drop out no grupo HCC) e K-3out(drop out no grupo SPE). RESULTADOS: Na coorte retrospectiva, 112 casos (59,3%) tinham CIR, 63 (33,3%) com HCC e 14 (7,4%) se enquadraram em SPE. Os tempos médios de avaliação até a inscrição em lista para TxH foram 194 dias (IC 95% 152-236), 36 dias (IC95% 21-50) e 98 dias (IC95% 0-308) para CIR, HCC e SPE, respectivamente (P <0,001). Dos 86 pacientes transplantados (K-out = 45,5%), 31 tinham CIR (K-1in = 27,7%), 44 HCC (K-2in = 69,8%) e 11 SPE (K-3in = 88,6%) (P <0,001). As taxas de drop out foram maiores em CIR (K-1out = 64,3%, K-2out = 30,2%, K-3out = 21,4%, P <0,001). O hazar ratio (HR) para TxH foi 85% (IC95% 1,35-2,55) maior em HCC do que CIR. Na coorte prospectiva, 110 dos 167 pacientes avaliados foram listados (K-in = 65,9%). Os tempos médios de avaliação foram de 783 dias (IC95% 330-1236), 52 dias (IC95% 17-87) e 184 dias (IC95% 19-349) para CIR, HCC e SPE, respectivamente (P <0,001). Em relação ao TxH, o K1-in foi 21,7%, K2-in, 76,4% e K3-in, 92,3 % (P <0,001). K-out foi 57,3% (63/110), K1-out = 50%, K2-out = 21,1% e K3-out = 3,84% (P <0,001). HR para TxH foi 329% superior em HCC do que CIR (HR = 4,29; IC95%: 2,74-6,72). CONCLUSÃO: Neste estudo, os pacientes com cirrose descompensada tiveram um tempo de avaliação para transplante significativamente maior que os outros grupos avaliados, bem como maior taxa de drop out em lista. A taxa de transplante foi significativamente menor nos pacientes com cirrose descompensada, demonstrando que as políticas de alocação de órgãos merecem ser revistas. / INTRODUCTION: Liver transplantation (LT) has been changing the course of serious, incapacitating and potentially fatal diseases becoming the treatment of choice when there is organ failure. AIM: To compare transplant, delisting, and survival rates between candidates with decompensated cirrhosis (CIR), special conditions (SPE), and hepatocellular carcinoma (HCC). METHODS: We carried out two studies with 358 patientes: a retrospective one (Aug 2008-Jul 2009, including 189 enlisted patients) and another prospective (Nov 2012-May 2014, with a follow-up period up to Nov 2015, including 169 LT candidates), comparing CIR, HCC, and SPE. The following variables were assessed: K-in (rates of waitlist entry – K-1in if CIR, K-2in if HCC, and K-3in if SPE); K-out (rate of LT); K-1out (drop-out in CIR); K-2out (drop-out in HCC); and K-3 out (drop-out in SPE). RESULTS: In the retrospective study, 112 cases (59.3%) were due to CIR, 63 (33.3%) to HCC, and 14 (7.4%) to SPE. The average time from selection to enlisting was 194 days (CI95% 152-236), 36 days (CI95% 21-50), and 98 days (CI95% 0-308) for CIR, HCC, and SPE, respectively (P<0.001). Of the 86 transplanted patients (K-out = 45.5%), 31 had CIR (K-1in = 27.7%), 44 HCC (K-2in = 69.8%), and 11 SPE (K-3in = 88.6%) (P<0.001). Drop-out rates were higher in CIR (K-1out = 64.3%, K-2out = 30.2%, K-3out = 21.4%, P<0.001). The hazar ratio (HR) for LT was 85% (CI95% 1.35-2.55) higher in HCC than CIR. In the prospective study, 110 out of 167 evaluated patients were enlisted (K-in = 65.9%). The average time from selection to enlisting was 783 days (CI95% 330-1236), 52 days (CI95% 17-87), and 184 days (CI95% 19-349) for CIR, HCC, and SPE, respectively (P<0.001). Regarding LT, K1-in was 21.7%, K2-in, 76.4%, and K3-in, 92.3% (P<0.001). K-out was 57.3% (63/110), K1-out = 50%, K2-out = 21.1%, and K3-out = 3.84% (P<0.001). HR for LT was 329% times higher in HCC than CIR (HR = 4.29; CI95% 2.74–6.72).CONCLUSION: In this study, patients with decompensated cirrhosis had a time evaluation for transplantation significantly higher than other evaluated groups as well as a higher rate of waiting list drop out. Transplant rate was significantly lower in patients with decompensated cirrhosis, demonstrating that organ allocation policies deserve to be reviewed.

INTRODUÇÃO: Na maioria dos casos, o carcinoma hepatocelular (CHC) acomete pacientes com cirrose hepática. O algoritmo do Barcelona Clinic Liver Cancer group (BCLC) considera função hepática, características tumorais e, estado geral do paciente para definir o tratamento. Entretanto, a aplicabilidade de um algoritmo terapêutico nem sempre é possível na prática clínica. Este estudo buscou avaliar a aderência às recomendações do BCLC para tratamento dos pacientes com CHC e analisar o impacto sobre a sobrevida nos diferentes estádios. MÉTODOS: Este estudo incluiu todos os pacientes encaminhados para o Instituto do Câncer do Estado de São Paulo para tratamento do CHC entre 2010 e 2012. Os pacientes (n = 364) foram classificados de acordo com as recomendações do BCLC. Se a terapia proposta não foi aplicada, o caso foi considerado como não aderente e as causas foram investigadas. A curva de sobrevida global foi estimada pelo método de sobrevida de Kaplan-Meier e comparada pela regressão de Cox. RESULTADOS: A porcentagem de aderência às recomendações do BCLC foi de 52% e variou entre os estádios: BCLC 0/A, 44%; BCLC B, 78%; BCLC C, 35%; e BCLC D, 67%. A taxa de sobrevida global, em um, dois e três anos, foi de 63, 45 e 33%, respectivamente. Nos pacientes aderentes do estádio BCLC 0/A, a sobrevida global foi significantemente melhor se comparada aos pacientes não aderentes (razão de risco [RR] = 0,19, intervalo de confiança [IC] 95%: 0,09-0,42; p < 0,001). Nos pacientes do estádio BCLC D, a taxa de sobrevida global foi pior em pacientes aderentes comparada aos pacientes não aderentes (RR = 4,0; IC 95%: 1,67-9,88; p < 0,001). Nenhuma diferença foi observada em pacientes do estádio BCLC B ou C classificados como aderentes ou não aderentes. CONCLUSÕES: A porcentagem de aderência às recomendações do BCLC na prática clínica é baixa e varia entre os estádios. A não aderência está associada a pior prognóstico, particularmente em pacientes com estádio precoce / INTRODUCTION: In most cases, hepatocellular carcinoma (HCC) affects patients with liver cirrhosis. Barcelona Clinic Liver Cancer group (BCLC) algorithm takes into consideration liver function, tumor variables and patients general status to guide therapy. However, the application of a therapeutic algorithm is not always feasible in clinical practice. The aim of this study was to assess the adherence of newly diagnosed hepatocellular carcinoma patients to the BCLC treatment guidelines, as well as examine the impact on survival in different stages. METHODS: This study included all patients referred to Instituto do Câncer do Estado de São Paulo for HCC therapy between 2010 and 2012. Patients (n = 364) were classified according to BCLC stage. If the proposed HCC therapy could not be applied, the case was considered to represent deviations from the recommended BCLC guideline. Causes of treatment deviations were investigated. The overall survival (OS) curves were estimated by the Kaplan-Meier survival method and compared by Cox regression. RESULTS: The overall percentage of adherence to BCLC guidelines was 52 % and varied among the disease stages: BCLC 0/A, 44 %; BCLC B, 78 %; BCLC C, 35 %; and BCLC D, 67 %. One-, two-, and three-year OS rates were 63, 45, and 33 %, respectively. In BCLC 0/A, adherent patients presented a significantly better OS compared to non-adherent patients (hazard ratio [HR] = 0.19, 95% confidence interval [CI]: 0.09-0.42; p < 0.001). In BCLC D, the OS rate was worse in adherent patients compared to non-adherent patients (HR = 4.0, 95% CI: 1.67-9.88; p < 0.001), whereas no differences were observed in BCLC stage B or C. CONCLUSIONS: The percentage of adherence to BCLC recommendations in clinical practice is low and varies among the clinical stages. Non-adherence is associated with a worse prognosis, particularly in early stage disease

Introdução: A Doença Hepática Gordurosa Não Alcoólica (DHGNA) tem sido considerada atualmente a forma mais comum de doença hepática no mundo ocidental, relacionada principalmente ao aumento da prevalência da obesidade.A DHGNA abrange um largo espectro de doença, desde casos de esteatose simples (EH) até esteato-hepatite não alcoólica (EHNA) e fibrose, podendo evoluir para cirrose e carcinoma hepatocelular (CHC). Embora se conheçam os fatores predisponentes para o desenvolvimento de EHNA, sua patogênese, assim como tratamento eficaz, permanecem pouco conhecidos.Os lípides, principalmente gorduras neutras, fosfolipídios e colesterol, são componentes fundamentais das células, assim como as proteínas e os hidratos de carbono. Embora aumentos marcantes de proteínas e hidratos de carbono não produzam quaisquer alterações macroscópicas no fígado, um acúmulo de gordura é prontamente reconhecido pela coloração amarelada e pelo aumento de volume do órgão. Várias dietas tem sido usadas em animais de laboratórios, principalmente camundongos e ratos no sentido de induzir esteatose, esteatohepatite e fibrose hepática, embora nenhuma delas consiga reproduzir na totalidade os componentes essenciais da doença humana. Não encontramos em nenhum trabalho da literatura a utilização da dieta deficiente em colina e hiperlipídica (DCH), por nós utilizada. Objetivo: O objetivo deste estudo foi avaliar o efeito de uma dieta deficiente em colina e hiperlipídica (DCH) para desenvolver esteatose hepática, esteatohepatite e fibrose hepática no rato. Métodos: A doença hepática foi induzida em ratos Sprague-Dawley machos pesando de 300 a 350 gramas, hospedados em gaiolas padrão e recebendo água à vontade. Foi usado dieta deficiente em colina e hiperlipídica (DCH). Os animais foram distribuídos por meio de tabela sequencial randomizada em dois grupos. No grupo 1 dez ratos receberam a dieta por 4 meses e no grupo 2 dez ratos receberam a dieta durante 8 meses. Após o tempo estipulado das dietas os animais foram anestesiados e sacrificados. Os fígados dos animais foram retirados, pesados e enviados para estudo histopatológico de acordo com os critérios estabelecidos por Kleiner. Os animais foram pesados no início do experimento e por ocasião do sacrifício. Resultados: o peso dos animais do grupo 1 (DCH - 4 meses) no início do experimento variou de 305 a 350 gramas com média de 329,9 gramas e no fim do experimento de 529 a 615 gramas com média de 579,2 gramas. O percentual de ganho de peso dos animais variou de 60 a 86,93% com média de 75,72%. O peso dos animais do grupo 2 (DCH - 8 meses) no início do experimento variou de 320 a 353 gramas com média de 339,4 gramas e no fim do experimento variou de 661,5 a 783 gramas com média de 705,6 gramas. O percentual do ganho de peso dos animais variou de 95,58 a 123,71% com média de 107,78%. O peso do fígado dos animais do grupo 1 no fim do experimento variou de 24,5 a 29,5 gramas. O percentual do peso do fígado em relação ao peso dos animais do grupo 1 variou de 4,16 a 5,54% com média de 4,75%. O peso do fígado dos animais do grupo 2 no fim do experimento variou de 31 gramas a 39 gramas com média de 33,8 gramas. O percentual do peso do fígado em relação ao peso dos animais do grupo 2 variou de 4,63% a 5,07%, com média de 4,78%. A análise histopatológica dos animais do grupo 1 demonstrou intensa balonização e esteatose microgoticular com inflamação lobular; o estadiamento de fibrose foi discreto nestes animais. O estudo histopatológico dos animais do grupo 2 revelou intensa esteatose microgoticular e balonização com inflamação lobular; alguns animais apresentaram esteatose macrogoticular. Os animais deste grupo 2 apresentaram fibrose com grande variação individual. Os animais de ambos os grupos experimentais desenvolveram esteatohepatite, pois apresentaram o índice de atividade da doença hepática gordurosa não alcoólica (NAS)>=5. A análise comparativa demonstrou não haver diferença estatística no valor do NAS entre os dois grupos experimentais. Em relação ao estadiamento da fibrose na esteatohepatite não alcoólica, os animais do grupo 2 apresentaram uma tendência de escores mais elevados do que os animais do grupo 1. No entanto não houve diferença estatística entre os grupos devido a grande variação individual (p=0,2755). Conclusões: A dieta DCH administrada durante 4 e 8 meses induziu nos ratos aumento considerável de peso corpóreo e do peso do fígado. Os animais do grupo 1 apresentaram intensa balonização, esteatose microgoticular e inflamação lobular com discreta fibrose hepática. Os animais do grupo 2 apresentaram intensa balonização, esteatose macro e microgoticular, inflamação lobular e maior grau de fibrose hepática, bem estabelecida com formação de colágeno e expansão fibrosa nos espaços vasculares. A dieta deficiente em colina e hiperlipídica (DCH) induziu no rato esteatose hepática e esteatohepatite com fibrose, servindo de modelo experimental para proporcionar melhor entendimento para procedimentos terapêuticos futuros desta importante patologia do fígado. / Introduction: Nonalcoholic Fatty Liver Disease (NAFLD) has currently been regarded as the most common form of liver disease in the western world, mainly related to the increased prevalence of obesity. NAFLD covers a broad spectrum of diseases, since cases of simple steatosis (HS) to steatohepatitis (EHNA) and fibrosis, and may evolve to cirrhosis and hepatocellular carcinoma (CHC). Although the predisposing factors for the development of EHNA are well established, there is little knowledge on its pathogenesis as well as the effective treatment options. Lipids, especially phospholipids, neutral fats and cholesterol, are fundamental cell components, as well as proteins and carbohydrates. Although striking increases of proteins and carbohydrates do not produce liver macroscopic changes, fat build up can be rapidly recognized by the yellow coloring and increased organ volume. Many diets have been used in laboratory animals, mainly rats although none of them can fully reproduce the fundamental components of the human disease. We could not find in the literature any study on the use of choline-deficient and hiperlipidic diet (CHD), used in the present study. Goal: The objective of this study was to evaluate the effect of a cholinedeficient and hiperlipidic diet (CHD) on the development of steatosis, steatohepatitis and hepatic fibrosis in rats. Methods: The liver disease was induced in male Sprague-Dawley rats, weighing from 300 to 350 grams, hosted in standard cages and receiving water ad libitum and fed with a choline-deficient and hiperlipidic diet (CHD). The experimental animals were distributed by means of a random sequential table in two groups. In Group 1 ten rats received the diet for four months and in Group 2 ten rats received the same diet for eight months. After these predetermined feeding time periods the animals were anesthetized and sacrificed. The animal livers were then removed, weighed and sent to histopathological study according to the criteria established by Kleiner. All the animals were weighed at the beginning of the experiment and by the time of the sacrifice. Results: The weight of Group 1 animals (4 months CHD) at the beginning of the experiment varied from 305 to 350 grams with an average of 329.9 grams and at the end of the experiment from 529 to 615 grams averaging 579.2 grams. The percentage of weight gain in animals of this group ranged from 60% to 86.93% with an average of 75.72%. The weight of Group 2 animals (8 months CHD) at the start of the experiment varied from 320 to 353 grams with an average of 339.4 grams and at the end of the experiment from the 661.5 to 783 g with an average of 705.6 grams. The percentage of weight gain in animals of this group varied from 95.58% to 123.71%, averaging 107.78%. The liver weight of Group 1 animals at the end of the experiment varied from 24.5 to 29.5 grams. The percentage of liver weight in relation to the animals\' weight, in Group 1, ranged from 4.16% to 5.54%, with an average of 4.75%. The liver weight of Group 2 animals at the end of the experiment varied from 31gramas to 39 grams, with an average of 33.8 gram. The percentage of liver weight in relation to the animals\' weight, in Group 2, ranged from 4.63% to 5.07%, with an average of 4.78%. Liver histopathological analysis in Group 1 animals showed marked ballooning degeneration and microgoticular steatosis with lobular inflammation; fibrosis staging was discreet in these animals. Liver histopathological study in animals of Group 2 showed an intense microgoticular steatosis and ballooning with lobular inflammation; some animals of this group presented macrogoticular steatosis. The amount of hepatic fibrosis in animals of this group was extremely variable. Animals of both experimental groups developed steatohepatitis, as they presented the activity index of nonalcoholic fatty liver disease (NAS) >= 5. Statistical analysis did not show a significant difference between the NAS values of the two experimental groups. Fibrosis staging analysis in non-alcoholic steatohepatitis showed a trend toward higher scores in animals of Group 2 as compared to Group 1 animals. However there was no statistical difference between the groups due to a large individual variation (p = 0.2755). Conclusions: CHD diet administered for four and eight months induced considerable increases in the rats\' body weight and liver weight. Animals in Group 1 showed intense liver ballooning, steatosis and lobular inflammation with discrete microgoticular fibrosis. Animals in Group 2 presented intense ballooning, steatosis macro and microgoticular, lobular inflammation and a higher degree of well-established hepatic fibrosis, with collagen formation with fibrotic expansion in vascular spaces. Choline-deficient and hiperlipidic diet (CHD) in the rat induced hepatic steatosis and steatohepatitis with fibrosis, representing an experimental model, which can provide a better knowledge for future therapeutic procedures of this important liver pathology.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-05T19:16:21Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2011-tese-DayseCeliaBarbosaLinsAroucha.pdf: 2035277 bytes, checksum: cb14694d5de87e893156d2d9926d27d2 (MD5) / Made available in DSpace on 2017-04-05T19:16:21Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2011-tese-DayseCeliaBarbosaLinsAroucha.pdf: 2035277 bytes, checksum: cb14694d5de87e893156d2d9926d27d2 (MD5) Previous issue date: 2011-12-13 / A infecção crônica pelo vírus da hepatite C (HCV) é uma das principais causas de doença hepática crônica, cirrose e carcinoma hepatocelular (CHC) em todo o mundo. A progressão para o CHC pode ser influenciada por fatores genéticos, como os polimorfismos de único nucleotídeo (SNPs) no promotor dos genes da interleucina 10 (IL-10) e do fator de necrose tumoral alfa (TNF-α), que regulam os níveis de expressão dessas citocinas. A interleucina 10 (IL-10) apresenta ação anti-inflamatória, antiangiogênica, possui um efeito modulatório na fibrogenese hepática e atua promovendo ou inibindo o desenvolvimento do tumor. Já o TNF-αé uma citocina pró-inflamatória que apresenta múltiplas funções no desenvolvimento e na progressão tanto in vitro como in vivodo câncer. Nossos objetivos foram determinar as freqüências gênicas e genotípicas (SNPs) no promotor dos genes da IL-10 e do TNF-α, verificar sua associação com atividade inflamatória e o grau de fibrose hepática, determinar os níveis séricos de IL-10 e do TNF-αe verificar sua associação com atividade inflamatória e o grau de fibrose hepática, em pacientes com hepatite C e o carcinoma hepatocelular causado pelo HCV(CHC-HCV). Foram avaliados 174pacientescom hepatite C, sendo 54 com fibrose leve e 65 com fibrose grave, além de 52 com CHC-HCV. A biópsia hepática foi classificada pelo score METAVIR e o diagnóstico do CHC-HCV seguiu os critérios da Barcelona Clinic Liver Cancer. Foi utilizada a PCR em tempo real para a investigação do SNPs e o ensaio imunoenzimático para dosagem sérica do IL-10 e TNF-αsegundo instruções de cada fabricante. As análises estatísticas utilizadas foram a univariada e multivariada. As concentrações séricas da IL-10 foram menores nos casos de CHC emais elevadas na fibrose leve (p<0, 0007),atividade inflamatória A1 (p<0, 0087) e A2 (p<0, 0079) nos pacientes com hepatite C.Já a concentração sérica do TNF-αfoi mais elevada nos pacientes com CHC-HCV quando comparadas aos pacientescom hepatite C com fibrose leve (p<0,0001), fibrose grave (p=0,004), atividade inflamatória leve(p=0.0004) e atividade inflamatória grave (p<0,001). Não houve diferença significativa entre as freqüências alélicas e genotípicas ou entre haplótipos e diplótipos do SNP no promotor do gene da IL-10 nos grupos avaliados. Por outro lado, as freqüências do alelo -308 G e do genótipo GG do SNP no promotor do gene do TNF-αforam significativamente maiores nos pacientes com CHC-HCV quando comparados aos pacientes com hepatite C que apresentavam fibrose levee atividade inflamatória leve. Em conclusão, houve uma relação inversa da concentração da IL-10 e TNF-αna hepatite C com fibrose grave e nos pacientes com CHC-HCV. Não foi encontrada relação com SNPs no promotor do gene da IL-10nos pacientes com hepatite C e CHC. Houve uma maior freqüência do alelo G e do genótipo GG do SNP no promotor do gene do TNF-αnos pacientes com CHC.O alelo GG foi fator de risco para a hepatite C com inflamação grave. / Chronic infection with hepatitis C virus (HCV) is the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. The progression to HCC may be influenced by genetic factors, such as single nucleotide polymorphisms (SNPs) in the promoter of the genes of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α), which regulate expression levels of these cytokines. The IL-10 is a multifunctional cytokine with anti-inflammatory and anti-angiogenics actions, has a modulator effect on hepatic fibrogenesis and acts by promoting or inhibiting tumor development. On the other hand, TNF-αis a pro-inflammatory cytokine with multiple roles, in vitroand in vivo, in the development and progression of cancer. Our aims were to determine the gene and genotypic frequencies of SNPs in the promoter of the IL-10 and TNF-αgenes,determine the serum levels of IL-10 and TNF-αand assess its association with inflammatory activity and liver fibrosis in hepatitis C patients and patients with hepatocellular carcinoma caused by HCV (HCC-HCV). We evaluated 119 patients with hepatitis C (54 with mild fibrosis and 65 with severe fibrosis) and 52 with HCC-HCV. Liver biopsy was classified by the METAVIR score and HCV-HCC diagnosis followed the criteria of the Barcelona Clinic Liver Cancer.We used real-time PCR for the SNPs investigation and enzyme-linked immunosorbent assay for serum IL-10and TNF-αfollowing manufacturer’s instructions. Serum levels of IL-10 were higher in mild fibrosis (p <0, 0007), A1 inflammatory activity (p <0.0087) and A2 (p <0.0079) in hepatitis C patients. The TNF-αserum levels were higher in patients with HCC-HCV compared to hepatitis C patients with mild fibrosis (p <0.0001), severe fibrosis (p = 0.004), mild inflammatory activity (p = 0.0004) and severe inflammatory activity (p <0.001). In the groups evaluated showed no significant differences in allelic or genotypic frequencies or in haplotypes or diplotypes at positions -1082 (G/A), -819 (C/T) and -592 (C/A) in IL-10 gene promoter. However, the frequencies of the -308 G allele and GG genotype of SNP in TNF-αgene promoter were significantly higher in patients with HCV-HCC compared to hepatitis C patients with mild fibrosis and mild inflammatory activity. In conclusion, we found that serum levels of IL-10 were higher in hepatitis C patients, whereas patients with HCV-HCC showed elevated TNF-αserum levels. The SNP in IL-10 gene promoter did not influence the degree of inflammatory activity and liver fibrosis, however, patients with HCV-HCC had a higher frequency of the G allele and GG genotype in TNF-αgene.

O presente estudo teve por objetivo estudar a participação da apoptose na carcinogênese hepatocelular, quantificando os corpos apoptóticos imunomarcados por caspase-3 clivada em amostras de carcinoma hepatocelular (CHC) em pacientes com ou sem cirrose, comparando também estes achados com amostras correspondentes de fígado não tumoral. Visou também à análise semi-quantitativa da imuno-expressão da proteína p53, Bax e Citocromo-C, relacionadas à via mitocondrial da apoptose em busca de eventuais relações com as variáveis clínicopatológicas dos carcinomas hepatocelulares. A análise comparativa da distribuição das diversas proteínas aqui estudadas foi ainda efetuada, com vistas à possível demonstração de sua interação no processo de apoptose em CHC. Amostras selecionadas de 79 casos de CHC foram distribuídas em micromatriz tecidual e submetidas a pesquisa imuno-histoquímica com amplificação por polímeros curtos de dextran ligados a peroxidase. IA foi maior nos CHC que nas amostras não-neoplásicas, mostrando ainda tendência a associação com o grau histológico do CHC .A imuno-expressão de p53 foi maior nos CHC em fígado cirrótico (CHC-C), em casos com invasão vascular, e nos graus histológicos altos. Houve maior imunoexpressão de citocromo c em CHC-C, sendo importante sua associação com p53. Bax mostrou apenas tendência a associação com o tamanho do CHC. Essas evidências contribuem para a compreensão da importância da via mitocondrial da apoptose mediada pela proteína p53 no CHC, destacando também prováveis diferenças do mecanismo carcinogenético na presença ou não de cirrose / This study aimed at the assesment of aspects of the role of apoptosis in hepatocellular carcinogenesis, quantifying apoptotic bodies immunomarked by cleaved caspase-3 in samples of hepatocellular carcinoma (HCC) in patients with or without cirrhosis, further comparing these findings to those from samples in non-tumoral areas of these livers. We also aimed herein to semiquantitate the immunoexpression of p53, Bax, Cytochrome-C, participants of the mitochondrial pathway of apoptosis, searching for possible relations with clinico-pathological variables in HCC. Samples from 79 cases of HCC were arranged in tissue microarrays were and submitted to immunohistochemical reaction with signal amplification achieved by the short-polymer-peroxidase system. Apoptotic index measured by immunoexpression of cleaved-caspase 3 was higher in HCC than in samples from non-neoplastic areas. p53 immunoexpression was higher in HCC occurring in cirrhotic livers, (HCC-C), in cases with vascular invasion and in higher histological grades. Cytochrome-c immunoexpression was also higher in HCC-C and, interestingly, was directly related to p53. Bax immunoreactivity showed only a trend for a relation with the size of HCC. The evidences from the present study further demonstrate the importance of p53-mediated pathway of apoptosis in HCC, and also point for possible differences in carcinogenesis in cirrhotic versus non-cirrhotic livers

A combinação de agentes quimiopreventivos com diferentes mecanismos de ação tem sido considerada uma estratégia promissora para a prevenção do câncer. Dentre os diversos compostos bioativos em alimentos, destacam-se a tributirina, um pró-fármaco do ácido butírico presente em laticínios e produzido pela fermentação de fibras dietéticas, e o óleo de linhaça, fonte de ácido alfa linolênico. Nesse contexto, foi avaliada a atividade quimiopreventiva de lipídios estruturados obtidos a partir da interesterificação enzimática de tributirina e óleo de linhaça durante a fase de promoção inicial da hepatocarcinogênese experimental. Ratos Wistar machos submetidos ao modelo do hepatócito resistente receberam diariamente, por via intragástrica (i.g), maltodextrina, óleo de linhaça, tributirina, a mistura não esterificada ou lipídios estruturados durante a fase de promoção inicial. O tratamento com lipídios estruturados demonstrou atividade quimiopreventiva comparável à da tributirina, mesmo resultando em menor concentração hepática de ácido butírico. Tanto a tributirina quanto os lipídios estruturados não inibiram a proliferação celular em lesões preneoplásicas, mas induziram a apoptose naquelas em remodelação. Os efeitos inibitórios da tributirina em fases iniciais da hepatocarcinogênese experimental estão relacionados ao aumento da acetilação de histonas e à modulação de processos de translocação nuclear da p53. No presente estudo, foi observado aumento substancial da razão nuclear/citoplasmática de p53 e importina-alfa em fígados de animais submetidos ao modelo e tratados com tributirina, mas não nos tratados com lipídios estruturados. Por outro lado, o tratamento com lipídios estruturados reduziu a expressão dos oncogenes Bcl2, Ccnd2, Pdgfa, Vegfa e aumentou a expressão dos genes supressores de tumor Cdh13, Fhit e Socs3. Assim, embora o potencial quimiopreventivo dos lipídios estruturados seja comparável ao da tributirina, os resultados sugerem que o novo composto não exibe atividade de HDACi, e que seus efeitos inibitórios na hepatocarcinogênese possam ser atribuídos à modulação da expressão de oncogenes e genes supressores de tumor. / Combination of chemopreventive agents with different mechanisms of action has been considered a promising strategy to cancer prevention. Among several bioactive food compounds, tributyrin, a butyric acid prodrug obtained from dairy products and dietetic fiber fermentation, and flax seed oil, a rich source of alpha linolenic acid have shown chemopreventive potential. Here, we evaluated the chemopreventive activity of structured lipids obtained by enzymatic interesterification of tributyrin and flax seed oil during the early promotion phase of experimental hepatocarcinogenesis. Male Wistar rats subjected to the resistant hepatocyte model were treated daily, i.g, with maltodextrin, flax seed oil, tributyrin, non-sterified blend, or structured lipids. Treatment structured lipids showed similar chemopreventive activity compared to tributyrin, even when structured lipids yielded lower concentrations of butyric in the liver. Tributyrin and structured lipids did not inhibit cell proliferation in preneoplastic lesions, but both of them induced apoptosis in remodeling preneoplastic lesions. In addition, histone acetylation and p21 restored expression tributyrin molecular mechanisms were related to modulation of p53 nuclear shuttling mechanisms. In the present study, it was observed a substantial increase in p53 nuclear/cytoplasmic ratio and importin-alpha in preneoplastic livers of tributyrin treated rats, but not in those treated with structured lipids. In contrast, treatment structured lipids downregulated expression of major oncogenes Bcl2, Ccnd2, Pdgfa, and Vegfa; and upregulated expression of critical tumor suppressor genes, Cdh13, Socs3 and Fhit. Hence, although structured lipids and tributyrin show similar chemopreventive potential, the results suggest that the new compound does not exhibit HDACi activity, and that its inhibitory effects may be attributed to the modulation of oncogenes and tumor suppressor genes expression.

O presente estudo avaliou materiais de 50 pacientes com cirrose hepática pelo vírus da hepatite C (VHC) que foram submetidos ao transplante hepático no Hospital das Clínicas da Faculdade de Medicina de São Paulo no período de 1993 a 2004, sendo divididos em dois grupos: Grupo 1 (33 pacientes com cirrose pelo VHC) e Grupo 2 (17 pacientes com cirrose pelo VHC com carcinoma hepatocelular). Nosso objetivo foi estudar a ocorrência da infecção oculta pelo VHB em pacientes com cirrose pelo VHC com ou sem CHC através do estudo molecular do genoma viral (DNA do VHB) no soro, tecido hepático tumoral e não tumoral pela utilização da técnica de Reação em Cadeia da Polimerase (PCR), pelos métodos in house e em tempo real. Todos os pacientes eram HBsAg negativos, possuíam soroteca e bloco de explante hepático em parafina, não apresentando concomitância com doenças hepáticas colestáticas, metabólicas e autoimunes. Foram avaliados os prontuários por um único pesquisador no sentido de coletar informações tais como: sexo, idade, dados de exames laboratoriais bioquímicos, sorológicos, ?fetoproteína e coagulação; além de dados clínicos tais como ascite e encefalopatia hepática para cálculos de índices prognósticos da cirrose (Child e MELD). Todo o material de explante hepático teve o Escore de Ishak e a Classificação das Sociedades Brasileiras de Patologia e Hepatologia para hepatites crônicas avaliados, assim como a Classificação de Edmondson e Steiner para os que apresentassem CHC. A técnica de PCR in house para detecção do DNA do VHB no soro e em tecido hepático tumoral e não tumoral apresentou negatividade em todas as amostras. Na técnica de PCR em tempo real apenas um caso do grupo 2 foi positivo no soro (sexo masculino, 66 anos, Anti-HBC total isolado e CHC); no tecido hepático tumoral no grupo 2 tivemos dois casos com resultados indeterminados e no tecido hepático não tumoral também do grupo 2, tivemos dois casos indeterminados. O grupo 1 não apresentou positividade para nenhuma das técnicas utilizadas. Concluímos que entre nossos pacientes com ou sem carcinoma hepatocelular associados à cirrose hepática pelo VHC, a infecção oculta pelo VHB foi muito baixa, provavelmente devido à baixa prevalência da infecção pelo VHB na nossa população / This study evaluated serum and liver tissue samples from 50 patients with liver cirrhosis due to hepatitis C virus (HVC) that underwent liver transplant at the Hospital das Clínicas - University of São Paulo School of Medicine during the period of 1993 to 2004, divided into two groups: Group 1 (33 cirrhotic patients due to HCV) and Group 2 (17 cirrhotic patients due to HCV with hepatocellular carcinoma - HCC). Our aim was to study the occurrence of occult HBV0 infection in cirrhotic patients due to HCV with or without HCC through the molecular study of HBV DNA in the serum, tumoral liver tissue and non tumoral liver tissue by the polymerase chain reaction (PCR) techniques using in house and real time PCR. All the patients were HBsAg negative, having previous serum samples frozen at -20ºC and liver tissue explanted in paraffin, without presenting concomitant cholestatic, metabolic and autoimmune liver diseases. The following variables were collected: gender, age, biochemical and coagulation laboratory tests and HBV serology (HBsAg, anti-HBc total, anti-HBs). Among the clinical data, ascites and encephalopathy were collected for the Child and MELD prognostic indexes. In the explanted liver tissue the Ishak\'s Score, The Brazilian Society of Pathology and Hepatology Classification for chronic hepatitis, and Edmondson and Steiner Classification for HCC were applied in the liver tissue. All samples with or without tumoral liver tissue and serum were negative for HBV DNA using in house PCR technique. By the real time PCR technique only one case from Group 2 was HBV DNA positive in serum (male, 66, isolated anti-HBc total positive and HCC). In the tumoral and non-tumoral liver tissues there were two indeterminated HBV DNA cases among Group 2 patients. All samples for Group 1 patients were negative for HBV DNA using both techniques. In conclusion, our study has shown the extremely low occult hepatitis B virus infection among the HCV cirrhotic patients with or without HCC, maybe due to the low HBV past infection among the Southeastern Brazilian population

Le carcinome hépatocellulaire (CHC) est une des principales causes de mortalité par cancer dans le monde. Dans environ 50% des tumeurs, on observe les signes d’une activation de la voie Wnt/β-caténine, causée par une mutation de l’exon 3 du gène CTNNB1 ou par stimulation du récepteur FRZD. Des études transcriptomiques du CHCs ont montré que ces deux modes d’activation étaient associés à des sous-types de tumeurs différents. Nous avons cherché à mieux comprendre les caractéristiques cliniques et le phénotype moléculaire de ces deux sous-types de CHCs. Dans un premier temps, nous avons fait le lien entre l'activation Wnt extracellulaire, un phénotype de cellules cancéreuses souches ou progénitrices et la présence de foyers de fibrose discrète intra-tumorale, observable par examen histopathologique, que nous avons appelés "nids fibreux". Nous avons également mis en évidence HAPLN1, une protéine de la matrice extracellulaire dont l’expression est stimulée par Wnt3a dans un modèle de cellules hépatiques progénitrices HepaRG, comme un nouveau marqueur d’agressivité du CHC. Ces résultats montrent une association entre l’activation Wnt extracellulaire et une agressivité tumorale passant par un remodelage matriciel. Dans un second temps, une Méta-analyse de données publiques de transcriptomique a permis de mettre évidence 4 sous-types de CHCs. La mutation CTNNB1, prédite par l’expression de 5 marqueurs par une méthode développée durant la thèse, était associée à un de ces sous-types et à un bon pronostic clinique. Nous avons également isolé un nouveau sous-type de CHC de bon pronostic exprimant un phénotype de tumeur différenciée et des signatures de métabolisme hépatique périportales. Ce sous-type a probablement été un facteur confondant dans les études précédentes mesurant l’association de la mutation CTNNB1 avec un bon pronostic. Enfin, nous avons mis en évidence une forte association négative entre la mutation CTNNB1 et l’inflammation ainsi que la fibrose tumorale dans trois cohortes indépendantes. Cet effet pourrait être provoqué par une inhibition de NF-κB par la β-caténine mutée, comme suggérée par des résultats préliminaires issue d’un modèle in vitro d’HepaRG mutés T41 stimulés par LPS. Nos résultats suggèrent donc que les deux modes d’activation de la voie Wnt/β-caténine sont associés à des mécanismes moléculaires, des profils d’expression, des phénotypes et des pronostics cliniques très différents. / Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Half of them show activation of Wnt/β-catenin pathway, caused by activating CTNNB1 exon 3 mutation of by stimulation of FRZD receptor. Transcriptomic based HCC classifications showed that this two types of activation were associated with distinct tumor subtypes. We tried to better understand the molecular phenotypes and the clinical features associated with these subtypes. In a first part, we linked extracellular Wnt activation with a stem/progenitor phenotype and with fibrous hotspot in HCC. Fibrous hotspot, which were called “fibrous nest”, can be detected by routine anatomic pathology analyses. We also showed that HAPLN1, an extracellular matrix protein induced by Wnt3a in progenitor HepaRG cells, was a new marker of stemness and bad outcome in HCC. Those results shows the associations between extracellular Wnt activation, extracellular matrix remodeling and tumor aggressiveness. In a second part, a transcriptome meta-analysis of 1133 HCCs highlighted 4 robust subclasses. CTNNB1 mutation, predicted by a 5-genes score based method, was associated with one of these subclasses and with good clinical features. We also highlighted a new subclass of CTNNB1 wild type HCCs associated with tumor differentiation, signatures of periportal metabolism and good outcome. This subclass was probably a confounding factor in survival studies comparing HCCs carrying mutant versus those carrying wild-type CTNNB1. Finally, we highlighted strong negative associations between CTNNB1 mutation and inflammation as well as tumor fibrosis in three independent cohorts. Preliminary results of in vitro HepaRG cells mutated for CTNNB1 in T41 and stimulated by LPS suggest an inhibitory effect of β-caténine on NF-κB. In conclusion, our results show that the two types of Wnt activation in HCC are associated with very distinct molecular phenotypes and clinical features.

L’infection par le virus de l’hépatite B (HBV) est problème majeur de santé publique mondial car, en dépit d’un vaccin efficace, les traitements curatifs actuels ne permettent pas l’élimination complète du virus. Comprendre les mécanismes de réplication du virus et son rôle dans la survenue du cancer hépatocellulaire (CHC) reste un enjeu majeur.Le rôle de la protéine HBx dans l’infection par HBV et l’oncogenèse viro-induite, reste mal connu, malgré un grand nombre de publications, car les fonctions décrites jusqu'alors sont limitées à des contextes d’études particuliers, souvent loin des conditions physiologiques.Mes travaux de thèse reposent sur l’utilisation de modèles d’études proches de la physiologie naturelle d’une infection par HBV, notamment des cellules primaires infectables in vitro. J’ai pu démontrer lors de mon étude que HBx est indispensable à la réplication de HBV, et qu’il agit essentiellement via son interaction avec DDB1 pour contrer la restriction du virus due au complexe SMC5/6, en induisant sa dégradation. Ce facteur de restriction permet de bloquer la transcription de l’ADN viral au niveau épigénétique. Ce nouveau rôle inattendu de SMC5/6 ouvre de nombreux axes de recherche, notamment sur les mécanismes de restriction des virus à ADN épisomal. SMC5/6 est connu pour son implication dans les voies de réparation de l’ADN : la dernière partie de ce manuscrit montre que sa dégradation dans les cellules infectées, altère ces mécanismes et sensibilise les cellules aux dommages à l’ADN, induits notamment par la radiothérapie. La présence de HBx dans les CHC pourrait ainsi s’avérer un atout pour le traitement du CHC / Hepatitis B virus (HBV) infection is a major health problem worldwide as (1) despite an effective preventive vaccine over 240 million individuals are chronically infected and (2) the actual viral suppressive treatments available do not eliminate viral DNA from cells. Thus, infected individuals are at a high risk of developing hepatocellular carcinoma (HCC) and understanding viral replication mechanisms and how it impacts on hepatocarcinogenesis is a major challenge.The role of the HBx protein, notably in viral replication and oncogenic processes, is the subject of many publications. However, many studies have often used non-physiological infection conditions. My thesis project has addressed these limitations by using cellular models, including primary human hepatocytes which can be infected by HBV, to investigate HBx’s role in these processes. I have shown that HBx is indispensable for HBV replication and that HBx associates with the infected cell’s DDB1/ E3 ubiquitin complex to target its Smc5/6 complex for degradation via the proteasome. These studies have identified that the Smc5/6 complex is a novel viral restriction factor that acts at an epigenetic level to block viral replication. This unexpected role of SMC5/6 has led to new research into the evolutionary conservation of restriction factors for episomal DNA viruses. As SMC5/6 is implicated in DNA Damage Repair (DDR), the last section of my thesis reports how SMC5/6 degradation in infected cells can sensitise cells to the cell killing effects of DNA damaging agents such as ionizing radiation and hydroxyurea. These results open-up possibilities for HCC treatment where HBx expression may be of therapeutic benefit

Le carcinome hépatocellulaire est un grand problème de santé publique et la troisième de mortalité lié au cancer dans le monde. Il a été démontré qu'au sein des tumeurs se trouve un petite population des cellules cancéreuses avec des propriétés de cellules souches cancéreuses. Elles sont responsables de l'initiation des tumeurs ainsi que de la récidive post-traitement et résistance aux thérapies. Peu de choses sont connues par rapport à la biologie de ces cellules mais l'identification des facteurs favorisant leur existence pourrait conduire vers des nouvelles pistes thérapeutiques.Nous avons trouvé que le facteurs de transcription p73 est surexprimé chez les patients dans les tumeurs du carcinome hépatocellulaire sous forme de deux types d'isoformes, les isoformes complets et les isoformes tronqués. Les isoformes complets sont des suppresseurs de tumeurs et corrèlent avec un meilleur taux de survie alors que les isoformes tronqués agissent comme dominants négatifs de ces premiers et favorisent la récidive post-chirurgie.Les résultats in vitro ont montré que les isoformes tronqués de p73 sont surexprimés dans les cellules souches cancéreuses du carcinome hépatocellulaire et favorisent leur emergence / Hepatocellular carcinoma (HCC) is a major public health problem, being the second most lethal cancer with an increasing incidence around the world. The only approved systemic drug is the multikinase inhibitor Sorafenib, which prolongs patients’ survival by only three months. HCC is refractory to known chemotherapeutic drugs and more than 50% of patients relapse after surgical tumor removal. These phenomena are thought to be due to the existence of a population of poorly differentiated cancer cells, largely known as liver cancer stem cells (CSCs). Recent studies revealed that CSCs activate similar pathways as normal stem cells. They are therefore highly resistant to therapies and are thought to be capable of self-renewal and generation of tumor’s heterogeneous cell mass. The understanding of mechanisms proper to liver CSCs should allow the development of innovative drugs with original mechanism of action against liver CSC, likely to improve patients’ outcome. However, the development of new therapies against HCC is penalized by the limited number of experimental models.According to these current challenges in the field of HCC research, my PhD thesis project covers three main axes: Development of novel models of disease (IMODI consortium)The Innovative Models of Disease (IMODI) consortium is mainly dedicated to the development of innovative experimental models for 7 different types of cancer. Our participation to the project concerned 3 main objectives i) development of HCC patient-derived xenografts ii) development of new HCC cell lines and iii) set up a cryoconservation method of primary human hepatocytes (PHHs) in the aim to employ them in humanizing murine livers. 30 patients planned for HCC tumor resection were recruited and their clinicopathological data were collected. Fresh tumor specimens were subcutaneously xenografted in immune-deficient mice and dissociated for in-vitro tumor cell culture. One tumor led to the development of a moderately differentiated HCC PDX model, as confirmed by histological characterization. Several studies showed the importance of PDX models in drug discovery as they recapitulate the drug-sensitivity patterns seen in patients from which they derive but very few models have been described in the literature for HCC. In vitro, primary HCC cells could be maintained in culture for a limited period of time, in average 30 days. No HCC cell lines developed due to cells entering replicative senescence, as previously described