Hepatitis C virus (HCV) infection globally affects 130-150 million people. It causes both acute and chronic infections. Due to the severe side effects and low success rates of interferon based treatments, which formed the standard treatment for HCV, the treatment paradigm shifted to direct acting antivirals (DAAs). DAAs have revolutionized the treatment of hepatitis C virus infection. Clinical trials with combinations of DAAs have recorded >90% response with shorter treatment durations and fewer side effects than earlier treatments involving IFN. Outside the controlled setting of a clinical trial, however, response rates with DAA combinations are much lower (<70%). DAAs can fail if HCV accumulates mutations that confer drug resistance. Interestingly, the pre-existence of mutant frequency in the virus appears not to influence treatment outcome. A better predictor for DAA treatment outcome is yet to be unravelled. Surprisingly, individuals who respond poorly to IFN appear to be more likely to fail DAA treatment. IFN is a generic antiviral that improves immune responses and is expected not to have any bearing on DAA treatment outcomes. Why individuals with poor IFN sensitivity fail DAA treatment remains a mystery. In a recent study of the IFN signalling network, HCV has been shown to compromise IFN activity. It induces bistability in the network leading to distinct phenotypic responses of cells to IFN exposure. In particular, individuals who respond poorly to IFN tend to have a higher percentage of cells that are refractory to IFN; these cells allow viral persistence despite IFN exposure. We hypothesized here that in such individuals, greater ongoing replication would allow increased development of resistance and thus lead to the failure of DAAs. We constructed a model of viral dynamics that accounts for the distinct phenotypic responses of cells to IFN, viral replication and mutation, and the development of resistance to DAAs. Our model predicted that although the relative prevalence of pre- existing mutants is unaffected by IFN sensitivity, in agreement with observations, the growth of drug resistant mutants is accelerated in individuals with poor IFN sensitivity. Based on a distribution of IFN sensitivity across individuals, our model accurately described clinical observations of the response rates to different current treatment protocols. With this model, we predict that the common strategy of increasing the genetic barrier by adding more drugs to the combination was not necessary to avert the development of drug resistance. Instead, an optimised increase in DAA dosage alone or DAA+PR or PR dosage depending on the patient’s IFN sensitivity could help achieve success.

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

Le VHC est une cause majeure de cancer du foie. Le traitement actuel est caractérisé par à un cout élevé, la présence de toxicité et l’émergence de résistance virale. Dans la 1ère partie de ma thèse, je me suis intéressé à l’entrée virale. L’entrée est nécessaire pour l’initiation ; la dissémination et le maintien de l’infection et représente ainsi une cible intéressante dans le développement de thérapies antivirales : CD81 et SRBI sont les 1ers facteurs décrits comme importants pour l’entrée : Nous avons confirmé leur rôle clé dans l’entrée et les étapes suivant l’entrée. De plus, nous avons montré leur rôle crucial dans la transmission cellule/cellule. Le VHC infecte principalement les hépatocytes, nous avons étudié en seconde partie de ma thèse le tropisme restreint du VHC aux hépatocytes. En définissant les facteurs essentiels à l’infection de cellules non hépatiques et en développant un modèle cellulaire afin d’identifier de nouveaux facteurs d’assemblage et de réplication du VHC. / HCV infection is the leading cause of chronic liver disease. The current SOC is still limited by high costs, toxicity and emergence of viral resistance. In the first part of my thesis we focused our workon viral entry. Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for the development of new antiviral therapies. CD81 and SR-BI are the first entry factors identified as important for HCV entry. In our work we confirmed their crucial role in entry, especially at the post-binding step. In addition we proved their key role in viral dissemination through the cell-cell transmission. As HCV mainly infects hepatocytes, we studied in the second part of my thesis, the restricted cellular tropism of HCV to hepatocytes and we defined the minimal host factors rendering non hepatic cell lines susceptible to HCV infection by the establishment of a powerful tool to identify new assembly and replication factors.

Introdução e Objetivos: Hipovitaminose D, definida como nível sérico de 25(OH)D insuficiente é considerada pandêmica em muitas populações ao redor do mundo e está associada a comorbidades em hepatite C, infecção por HIV e coinfecção HIV/HCV. Os objetivos deste estudo são: 1) comparar a prevalência de deficiência de vitamina D (DVD) caracterizada por nível sérico de 25(OH)D < 20 ng/mL, entre pacientes monoinfectados pelo HCV, monoinfectados pelo HIV, coinfectados HIV/HCV e participantes do grupocontrole; 2) identificar fatores de risco específicos associados com DVD na população estudada. Pacientes e Métodos: Foram coletados dados clínicos e demográficos, 25(OH)D sérica, testes de função hepática e perfil metabólico durante os meses de inverno de 129 pacientes HCV monoinfectados, 118 pacientes HIV monoinfectados e 53 pacientes coinfectados HIV/HCV tratados em centros de referência na cidade de São Paulo, bem como, em 122 indivíduos saudáveis de um grupo-controle formado de pessoas não infectadas por HIV, HCV ou HBV, sem uso de suplementos de vitamina D. Resultados: A prevalência de deficiência de vitamina D ajustada por sexo, idade ( = 50), cor de pele (branco vs não branco), índice de massa corporal ( = 25), colesterol total (= 200), fração HDL colesterol ( = 40 em homens = 50 em mulheres), triglicérides (= 150), glicemia ( = 110), uso de efavirenz (sim vs não), uso de tenofovir (sim vs não) e índice HOMA-IR, foi menor no grupo HCV do que no controle e no grupo HIV (p < 0.001). Em todos os grupos, a razão de chance de DVD aumenta 1.21 [IC95%(1.01; 1.44) p=0.026] para cada ponto de aumento do índice HOMA. Efavirenz também esteve associado com maior razão de chance de DVD [3.49(IC95% 1.14-10.67) p=0.028]. Análise por regressão logística simples foi aplicada para avaliar fatores de risco associados a DVD dentro de cada grupo. Nesta análise, resultaram com associação significativa o sexo masculino, com uma menor razão de chance para DVD [RC 0,42(IC95% 0,18 - 0,96) p = 0,04] no grupo-controle e no grupo HCV [RC 0,42(IC95% 0,2 - 0,88) p = 0,02]; ainda no grupo HCV houve associação significativa entre DVD e HOMA-IR elevado [RC 5,59(IC 95% 1,37 - 22,8) p = 0,02]; e, no grupo HIV, os indivíduos que apresentaram nadir histórico de CD4 maior que 200 células/mm3 tiveram menos chance de DVD [RC 0,41 (IC95% 0,18 - 0,95) p = 0,04]. Conclusão: Uma alta prevalência de DVD foi observada em toda a população estudada, incluindo o grupo-controle, sugerindo que a apresentação de infecção por HIV e/ou HCV por si só não aumenta as chances de DVD. Por outro lado, o incremento do índice HOMA e o uso de efavirenz se destacaram como fatores de risco nesta população. Estes achados ressaltam a importância da associação da deficiência de vitamina D com outras duas condições; a resistência à insulina e o uso de terapia antirretroviral para o HIV, os quais, isoladamente ou em combinação, podem aumentar a incidência de comorbidades como o diabetes do tipo 2 / Background and Aims: Hypovitaminosis D, defined as insufficient serum level of 25(OH)D, is considered pandemic in many populations worldwide and is associated with co-morbidities in hepatitis C, HIV and HIV/HCV co-infection. The aim of this study is to 1) compare the prevalence of 25-hydroxyvitamin D deficiency (VDD), defined as serum levels of 25(OH)D < 20 ng/mL, among HCV mono-infected, HIV mono-infected, HIV/HCV co-infected patients and control participants and 2) identify specific risk factors associated with VDD in each group. Patients and Methods: We collected demographic and clinical data, serum 25-hydroxyvitamin D, liver function parameters and metabolic profiles on 129 HCV mono-infected, 118 HIV mono-infected and 53 HIV/HCV co-infected patients treated at reference centers in São Paulo (Brazil) as well as on 122 volunteer controls, not infected by HIV, HCV, HBV or taking vitamin D supplements. Results: VDD prevalence adjusted for sex, age ( = 50), skin color (white vs not white), body mass index ( = 25), total cholesterol (= 200), HDL cholesterol ( = 40 in men and = 50 in women), triglycerides ( = 150), glycemia ( = 110), use of Efavirenz - EFV (yes vs no), use of Tenofovir -TDV (yes vs no) and HOMA-IR was lower in HCV group than control and HIV groups (p < 0.001). In all groups, adjusted odds of VDD increases by 1.21 [CI95% (1.01-1.44)] for each unit increase of HOMA-IR. Antirretroviral therapy regimens containing efavirenz were also associated to higher odds of VDD 3.49 [CI95% (1.14-10.67) p=0.028]. Logistic regression was applied to analyze risk factors associated to VDD within each group. In this analysis male sex resulted significantly associated to lower chance of VDD [OR 0,42(CI95% 0,18 - 0,96) p = 0,04] in control group and in HCV group [RC 0,42(CI95% 0,2 - 0,88) p = 0,02]; still in HCV group, elevated HOMA-IR was significantly associated to VDD [OR 5,59(CI 95% 1,37 - 22,8) p = 0,02]; and in HIV group, individuals presenting CD4 nadir higher than 200 cells/mm3 had less chance of VDD [OR 0,41 (IC95% 0,18 - 0,95) p = 0,04]. Conclusion: High prevalence of VDD was observed across all studied population, including control group, suggesting that being infected with HIV and/or HCV per se does not increase the chance of VDD. Otherwise, VDD was positively associated with HOMA-IR increase for controls and infected patients. It is also associated to use of Efavirenz in HIV/HCV patients. This finding highlights the relevance of vitamin D deficiency association with two other conditions; insulin resistance and antiretroviral therapy, which isolated or in combination, may contribute to the incidence of comorbidities, as Type 2 diabetes mellitus

Estima-se que 3% da população mundial esteja infectada pelo vírus da hepatite C (HCV). O HCV tem como alvo o tecido hepático e a maioria dos pacientes infectados desenvolvem infecção crônica. Nos últimos anos, estudos in vitro têm demonstrado interações entre o miRNA-122 (miR-122) da célula hospedeira e dois sítios localizados na região 5\' UTR do genoma do vírus da hepatite C (HCV), os quais são essenciais ao processo de replicação viral. O miR-122 é altamente expresso no fígado, onde atua na regulação do metabolismo de lipídios juntamente com outro miRNA, o miRNA-33a (miR-33a), porém, o mecanismo envolvido nesta regulação ainda é pouco conhecido. Sabe-se que a infecção pelo HCV altera a expressão de genes envolvidos na biossíntese e transporte de lipídios, resultando na estimulação do metabolismo de lipídios e criando um ambiente favorável para sua replicação. Neste contexto os objetivos deste trabalho foram avaliar a expressão de miR-33a e miR-122 em indivíduos cronicamente infectados pelo HCV-1 e HCV-3 em amostras obtidas antes do início da terapia. Os miRNAs foram isolados a partir de amostras de sangue periférico e de tecido hepático. A quantificação da expressão relativa de ambos miRNAs foi pela técnica de PCR em tempo real. Os níveis de miR-33a no sangue periférico foram mais elevados do que no tecido hepático em indivíduos infectados pelo HCV-1(p < 0,0001) e HCV-3 (p=0,0025). Observou-se uma correlação inversa entre os níveis de miR-33a no sangue periférico e tecido hepático dos indivíduos infectados pelo HCV-1 (r=-0,281, p=0,039) e correlação positiva para os indivíduos infectados pelo HCV-3 (r=0,9286, p < 0,0001). Correlação inversa entre os níveis hepáticos de miR-33a com o nível sérico de insulina (r=-0,371, p =0,005) nos indivíduos infectados pelo HCV-1 e correlação positiva entre os níveis no sangue periférico com os níveis séricos de GGT (r=0,553, p=0,049) foram observadas. Em relação ao miR-122, de maneira geral o nível hepático foi mais elevado do que o sérico (p < 0,0001). Entretanto, o nível hepático de miR-122 em indivíduos infectados pelo HCV-3 foi maior quando comparado aos infectados pelo HCV-1 (6,22 vezes, p < 0,001). Uma correlação inversa entre os níveis séricos de ApoA-II e os níveis de expressão de miR-122 no sangue (r=-0,330; p=0,014) e tecido hepático (r=-0,311; p=0,020) foi observada nos pacientes infectados pelo HCV-1. Os pacientes infectados pelo HCV- 3 mostraram correlação positiva entre os níveis hepáticos de miR-122 e os níveis de HDL (r=0,412, p=0,036) e insulina (r=0,478, p=0,044). O miR-33a e o miR-122 atuam regulando genes que controlam o metabolismo dos lipídios no fígado. Até o presente momento, não existem relatos que associem a expressão do miR-33a e do miR-122 com o perfil lipídico na infecção pelo HCV. Além disso, o acúmulo de lipídio (esteatose) intensamente descrito na infecção pelo HCV-3 pode sugerir interação diferenciada desse genótipo com os mecanismos envolvidos na regulação do metabolismo lipídico, envolvendo o miR-33a e miR-122 / The prevalence of infection by hepatitis C virus (HCV) is about 3% of the world population. HCV targets the liver tissue and the majority of infected patients develop chronic infection. In recent years, in vitro studies have demonstrated interactions between miRNA-122 (miR-122) the host cell to two places located in the 5\' untranslated region of the HCV genome which are essential for virus replication process. miR-122 is highly expressed in the liver, which has been implicated as a fatty acid metabolism regulator. Another mine has also been described as a key regulator of lipid metabolism, miRNA-33a (miR-33a), however, the mechanisms involved in this regulation are still little known. It is known that HCV infection changes the expression of genes involved in the biosynthesis and transport of lipids, resulting in stimulation of the lipid metabolism and creating a favorable environment for replication of the virus. To our knowledge, there are no reports linking the expression of miR-33a with lipid profile in HCV infection. In this context the objectives of this study were to evaluate the expression of miR-33a and miR-122 in chronically infected individuals with HCV-1 and HCV-3 in samples obtained prior to initiation of therapy. MiRNAs were isolated from peripheral blood samples and liver tissue. The quantification of relative expression of both miRNAs was by PCR in real time. MiR-33a levels in peripheral blood were higher than in liver tissue in patients infected with HCV-1 (p < 0.0001) and HCV-3 (p=0.0025). Levels in the peripheral blood of miR-33a were lower in patients infected with HCV-3 (p=0.0169). There was an inverse correlation between hepatic levels of miR-33a with serum insulin levels (p=0.005) in individuals infected with HCV-1 and a positive correlation between the levels in the peripheral blood serum levels of GGT (p=0.049). Hepatic levels of miR-122 were higher than the levels in the peripheral blood of individuals infected by HCV-1 and HCV-3 (p < 0.0001). Hepatic miR-122 levels were higher in patients infected with HCV-3 than those infected with HCV-1 (6.22 times, p < 0.001). There was a positive correlation between miR-122 levels in the blood and liver tissue of patients infected with HCV-1 (r=0.302, p=0.026). An inverse correlation between serum ApoA-II was observed in these patients the levels of expression of miR-122 in blood (r=-0.330; p =0.014) and liver tissue (r=-0.311; p=0.020). Patients infected with HCV-3 showed a positive correlation between hepatic miR-122 levels to HDL levels (r=0.412, p=0.036) and insulin levels (r=0.478, p=0.044). The miR-33a and miR-122 act by regulating genes that control lipid metabolism in the liver. The different interactions with lipid metabolism exerted by HCV-3 may explain why his relationship with the miR-33a and miR-122 was different when compared with HCV-1

A doença hepática crônica causada pelo vírus da hepatite C (HCV) tornou-se, nos últimos anos, uma das principais comorbidades dos pacientes portadores do vírus da imunodeficiência humana (HIV) nos países desenvolvidos. Os pacientes coinfectados com HIV/HCV apresentam uma progressão mais rápida para a cirrose e as suas complicações que os pacientes monoinfectados com HCV. Embora os mecanismos responsáveis por esta evolução não estejam totalmente esclarecidos, a expressão da molécula de HLA-G, um HLA de classe Ib não clássico, que tem propriedades bem reconhecidas na regulação negativa da resposta imune, pode estar relacionada à progressão da doença hepática. Os objetivos deste trabalho foram analisar o perfil de expressão de HLA-G em tecido hepático de pacientes coinfectados HIV/HCV e identificar possíveis variáveis do hospedeiro, do HCV e do HIV que possam estar relacionadas com a expressão de HLA-G na biópsia hepática. Para isso, 57 amostras de biópsia hepática de pacientes coinfectados com HIV/HCV, nas quais a imuno-histoquímica para HLA-G foi realizada, foram analisadas retrospectivamente quanto à expressão desta molécula no tecido hepático. Avaliaram-se também outras características histopatológicas da biópsia como grau de fibrose, atividade inflamatória, deposição de ferro e gordura. Determinou-se o polimorfismo de inserção ou deleção de 14 pares de bases da região 3` não traduzida do exon 8 do gene do HLA-G, que está relacionada com a produção de RNA-mensageiro, em 43 destes pacientes, além do polimorfismo de IL-28B, relacionado com a resposta ao tratamento do HCV, em 44 deles. Características bioquímicas e virológicas, tanto do HIV quanto do HCV também foram avaliadas. O genótipo 1 do HCV foi o mais prevalente (87,75%), especialmente o subgenótipo 1a (60%). A expressão do HLA-G foi observada em 38 (66,7%) amostras de fígado, e foi mais frequente em estágios moderados e severos de fibrose do que em estágios mais leves (94,1% x 55%, P < 0,01). Não houve relação entre a expressão do HLA-G e os outros parâmetros estudados. Embora a progressão para a cirrose no contexto da coinfecção por HIV/ HCV seja um processo complexo, modulado por muitos factores, a associação da intensidade de fibrose com a expressão do HLA-G pode indicar que a expressão desta proteína desempenha um importante papel nos mecanismos que contribuem para a progressão da doença, por meio da regulação negativa da resposta imune contra o HCV na coinfecção pelo HIV. / Chronic liver disease induced by hepatitis C virus (HCV) infection has recently become one of the most common comorbidities in patients who are infected with the human immunodeficiency virus (HIV) in developed countries. HIV/HCV coinfected patients show faster progression to cirrhosis and its complications than the HCV monoinfected patients. Even though the responsible mechanisms for this evolution have not been entirely clarified yet, the expression of the HLA-G molecule, a HLA from the non-classic Ib class, with well-known properties of negatively regulating the immune response, may be related to the liver disease progression. The aims of the present work were to analyze the HLA-G expression profile in the liver micro ambience of HIV/HCV coinfected patients and to identify possible host factors, HIV or HCV, that may be related to the HLA-G expression on the liver biopsy. For this purpose, 57 liver biopsies of HIV/HCV coinfect patients, in which immunohistochemistry for HLA-G had been performed, were retrospectively analyzed according the HLA-G expression on the hepatic tissue. Other histopathological features in the liver biopsies, such as fibrosis degree, inflammatory activity, iron deposition and fat were also evaluated. The polymorphism of insertion or deletion in 14-base pairs of the 3`non-translated region of exon 8 of the HLA-G gene, which is related to the production of HLA-G messenger RNA, was evaluated in 43 of the patients. Also, the polymorphism of IL-28B, related to the response to HCV treatment, was evaluated in 44 of them. Biochemical and virological features of HIV and HCV were also evaluated. The HCV genotype 1 was the most prevalent (87.75%), especially the subgenotype 1a (60%). The expression of HLA-G was observed in 38 (66.7%) samples of the liver biopsies, and it was most frequent in moderate and severe stages of fibrosis than in the mild stages (94.1% x 55%, P < 0.01). There was no established relationship between HLA-G and other parameters studied. Although the progression to cirrhosis in the context of HIV/HCV coinfection is a complex process modulated by many factors, the association of HLA-G expression with the intensity of the liver fibrosis may indicate the protein expression play an important role in the mechanisms that contribute to the progression of the disease, through the negative regulation of the immune response against HCV setting of a coinfection with HIV.

Zwischen August 1978 und März 1979 wurden in der DDR durch viruskontaminiertes Anti-D-IgG 2867 Frauen mit HCV-Genotyp 1b infiziert, von denen 427 Frauen aus dem Leipziger Raum stammen. Diese Arbeit untersucht nach über 30 Jahren HCV-Infektion den natürlichen HCV-Verlauf von 356 der 427 Frauen aus dem Leipziger Raum. Ziel dieser Untersuchung ist herauszufinden, ob eine HCV-Infektion mit einer erhöhten Leberzirrhoserate bzw. erhöhten Mortalitätsrate einhergeht. Dabei stellen die 356 Frauen in dieser Untersuchung die Gesamtkohorte dar. In die Auswertung der Teilkohorte konnten 181 der 356 Frauen eingeschlossen werden, die im Zeitraum von 2005-2011 mindestens einmal untersucht und von denen aktuelle klinische und laborchemische Parameter erhoben wurden. Patientinnen mit HC-Viruspersistenz wurden z. T. mehrfach untersucht. Von der Gesamtkohorte wurden alle vorhandenen Leberbiopsien, serologische und molekularvirologische Daten wie anti-HCV, HCV-PCR und Viruslast, Leberzirrhose-Eintritt, Todesfälle, Formen der akuten Hepatitis-C-Infektion (ikterisch, anikterisch, asymptomatisch) erfasst. Die bestehenden Daten der Gesamtkohorte waren Grundlage für die Berechnung der Gesamt-Leberzirrhoserate und Gesamtmortalität sowie für die Erstellung der Kaplan-Meier-Kurven und Überlebenstabellen. Dabei wurde in den Überlebenskurven der Eintritt einer Leberzirrhose bzw. der Tod als Ereignis definiert. Für die Auswertung der Teilkohorte (n=181/356) wurden zusätzlich laborchemische Parameter (u. a. Transaminasen), Komorbiditäten (BMI, Diabetes mellitus, Steatosis hepatis), Koinfektionen, FibroScan-Werte, IL28 rs 12979860 Polymorphismus bzw. durch Anamnesebögen bestehender Alkoholkonsum, Medikation, Begleiterkrankungen bzw. subjektive Beschwerden, ermittelt. Bei mehreren Alanin-Aminotransferase-Werten (ALAT-Werten) wurde der Verlauf über fünf Jahre klassifiziert als ständig normwertig, fluktuierend, ständig erhöht und erhöht aufgrund hepatotoxische Einflüsse, die nicht allein HCV-bedingt sind (z. B. durch Alkoholkonsum, Steatosis hepatis, Medikation, Cholestase). In dieser Untersuchung wurden drei Gruppen untereinander verglichen. Patientinnen mit einem akut selbstlimitierenden Verlauf bzw. Patientinnen ohne HCV-Nachweis stellen die Kontrollgruppe (HCV-RNA negative Gruppe) dar, in der Annahme, dass die nur kurz verweilende Hepatitis-C-Infektion bei den akut selbstlimitierenden Verläufen keinen wesentlichen Einfluss auf die Überlebensrate hat. Die HCV-RNA positive Gruppe setzt sich aus therapienaiven HCV-Infizierten und antiviral therapierten HCV-Infizierten zusammen, die keine sustained virological response (SVR) erreicht haben, sogenannte Non-Responder. Diese HCV-RNA positive Gruppe soll das Risiko des weitgehend natürlichen Hepatitis-C-Verlaufes darstellen. Zu der Gruppe mit SVR zählen Patientinnen, die sechs Monate nach einer erfolgreichen antiviralen Therapie weiterhin HCV-PCR negativ sind. Patientinnen mit einer SVR wurden gesondert betrachtet, um einen zu starken Einfluss auf den natürlichen HCV-Verlauf auszuschließen. Dabei war die antivirale Ansprechrate mit Erreichen einer SVR in der Teilkohorte mit 60,1% höher als vorbeschrieben. Eine akute Hepatitis-C konnte bei 279/356 Patientinnen eruiert werden, von denen 82/270 (29,4%) einen ikterischen Verlauf aufwiesen. Dabei wird ein homozygoter CC-Polymorphismus mit einem ikterischen Verlauf und mit einer spontanen Viruselimination assoziiert. Unter Ausschluss der Patientinnen ohne HCV-Nachweis entwickelten 173/329 (53%) eine chronische Hepatitis-C. Das durchschnittliche Alter bei Infektion betrug 24 (16-38) Jahre und zum Zeitpunkt der Studie 54 (46-71) Jahre. Von der Teilkohorte waren 154/181 Frauen (85,1%) weiterhin anti-HCV positiv und bei 59/181 Frauen (32,6%) wurde HCV-RNA detektiert. Die Hälfte der Frauen (92/181; 50,8%) hatten permanent normwertige ALAT-Werte und bei 51/181 Frauen (28,2%) wurden fluktuierende ALAT-Werte ermittelt. Nur 38/181 Frauen (21%) wiesen permanent erhöhte ALAT-Werte auf, die v. a. bei HCV-RNA positiven Patientinnen nachgewiesen wurden, welche mit einer erhöhten Leberzirrhoserate assoziiert werden (p=0,001). Ein weiterer Leberzirrhose-assoziierender-Kofaktor ist ein bestehender Diabetes mellitus (p=0,013). Zusätzlich wird ein Diabetes mellitus mit einem vermehrten Auftreten von Steatosis hepatis (p=0,005) in Verbindung gebracht. Ein Zusammenhang zwischen Steatosis hepatis und erhöhte Leberzirrhoserate besteht hingegen nicht (p=0,839). In allen Gruppen ist mit steigendem Alter ein Zuwachs an Adipositas (32%), Diabetes mellitus (7,7%) und Steatosis hepatis (25,4%) vorhanden. Jedoch konnte bisher kein wesentlicher Einfluss auf die Leberzirrhoserate bzw. Mortalitätsrate bei HCV-RNA positiven Patientinnen nachgewiesen werden. Dennoch sollten Patienten mit einer chronischen HCV-Infektion auf eine Diabetes mellitus Manifestation getestet werden, um ggf. eine antivirale Therapie bei erhöhtem Leberzirrhose-Risiko zu beginnen. In künftigen Studien sollten diese Parameter mehr berücksichtigt werden, um einen möglichen Einfluss auf die Leberzirrhoserate feststellen zu können. Erwartungsgemäß weisen HCV-RNA positive Patientinnen einen höheren intrahepatischen Inflammations- und Fibrosegrad auf als HCV-RNA negative Patientinnen. Vor allem ist ein starker Anstieg an Leberzirrhosen bei den HCV-RNA positiven Frauen ab einem Lebensalter über 50 Jahre zu erkennen. Dennoch ist die Gesamtzirrhoserate 32 Jahre nach HCV-Infektion mit nur 6,45% (22/341) erstaunlich niedrig. Eine Erklärung ist, dass durch den exakt bekannten Infektionszeitpunkt dieser Untersuchung das gesamte HCV-Spektrum (Genotyp 1b), inklusive aller akut selbstlimitierenden Verläufe, Patientinnen ohne HCV-Nachweis und Patientinnen mit einer chronischen Hepatitis-C, erfasst wurde. Somit konnte eine einseitige Patientenselektion zu Gunsten fortgeschrittener Leberfibrosen von chronischen Hepatitis-C-Patientinnen vermieden werden. Ein weiterer Vorteil liegt in dem jungen Infektionsalter mit resultierenden geringen Komorbiditäten, die den natürlichen HCV-Verlauf beeinflussen könnten. Dennoch ist mit zunehmender Studienlänge eine steigende Unterrepräsentation an Patientinnen mit akut selbstlimitierenden Verläufen zu verzeichnen, die im Gegensatz zu den Patientinnen mit chronischer Hepatitis, bei fehlender Symptomatik nicht mehr kontinuierlich den Arzt konsultieren. Dadurch ist in der Teilkohorte der Anteil an fortgeschrittenen Leberzirrhosen mit 12/181 (6,6%) Präzirrhosen und 16/181 (8,8%) Leberzirrhosen erhöht. Dabei sind sieben Frauen mit einer Präzirrhose und 11 Frauen mit einer Leberzirrhose HCV-RNA positiv. Werden die Patientinnen mit einer SVR ausgeschlossen, erhöht sich die Zirrhoserate in der Teilkohorte auf 9,8% und in der Gesamtkohorte auf 6,9%. Die „wahre“ Leberzirrhoserate liegt womöglich zwischen diesen Prozentsätzen. Überraschenderweise besteht bisher keine erhöhte Mortalität bei HCV-RNA positiven Patientinnen im Vergleich zu HCV-RNA negativen Patientinnen, obwohl die Leberzirrhoserate bei den HCV-RNA positiven Patientinnen signifikant erhöht ist (Log-Rang: p=0,001). Insgesamt sind 14 von 349 Frauen (4%) verstorben, von denen acht nicht-HCV-assoziiert waren. Bedingt durch den verstärkten Einsatz von Dualtherapien mit Peg-Interferon und Ribavirin nimmt der Anteil chronisch HCV-Infizierter zu Gunsten der Patientinnen mit SVR kontinuierlich ab. Dennoch sind Leberzirrhosen und Todesfälle in der Gruppe mit SVR zu verzeichnen, die als Spätkomplikation einer durchlebten HCV-Infektion gewertet werden sollten, um eine weitere Patientenselektionen zu verhindern. Durch eine SVR sind normwertige Transaminasen bzw. regrediente Inflammations- bzw. Fibrosegrade zu erreichen. So haben von der Teilkohorte 36/48 (75%) der Patientinnen mit SVR normwertige ALAT-Werte. Die Zirrhoserate von Patientinnen mit SVR liegt 32 Jahre nach HCV-Infektion mit 11,5% signifikant unter der von HCV-RNA positiven Patientinnen mit 30%. Dennoch lässt sich eine erhöhte Überlebenswahrscheinlichkeit von Patientinnen mit SVR gegenüber HCV-RNA positiven Patientinnen momentan nicht belegen. Die nächsten 10 bis 20 Jahre werden eine richtungsweisende Aussage über die Überlebenskurven zeigen. Dafür sind weitere Langzeitstudien nötig. Jedoch wird es zukünftig immer schwieriger werden die Kohorte weiterzuverfolgen. Der Grund liegt in der anhaltenden Abnahme der untersuchten Patientinnen, bedingt durch Umzug, Namens- oder Arztwechsel. Zusammenfassend ist nach über 30 Jahren HCV-Infektion nur ein mäßiger Anstieg an Leberzirrhosen in einer weiblichen Population mit jungem Infektionsalter zu verzeichnen. Jedoch sollte bei zu erwartendem stärkeren Anstieg an Leberzirrhosen, bei HCV-RNA positiven Patientinnen ab einem Alter über 50, eine frühzeitige antivirale Therapie erwogen werden.

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Hepatitis C Virus (HCV) infection represents a worldwide pandemic and is currently the leading cause of cirrhosis and liver transplantation. After transplantation recurrence is almost universal with 96% of patients testing positive for viral RNA and exhibiting histological evidence of infection within the first year. Type I interferons (IFN) and interferon inducible genes are responsible for the innate antiviral state and single nucleotide polymorphisms (SNPs) within these genes may affect the patients ability to respond post-transplantation. We hypothesize the elucidation of associations between SNPs in Type-I Interferon and Interferon inducible genes and HCV recurrence post-liver transplantation might help to identify HCV patients with different prognosis and improve liver transplant recipient selection. 100 HCV positive patients were genotyped using Allelic Discrimination on an ABI Prism 7700 sequence detector (Applied Biosystems) for SNPs in IFNB1, OAS-1, and ISG-15 to establish a relationship between SNPs and clinical complications post-transplantation. Quantitative real-time polymerase chain reaction (QPCR) was also run to determine the relationship between SNPs or disease state and the level of RNA expression. Results were collected and analyzed using Fishers exact test, Kaplan-Meir method, and the log-rank test. Results obtained indicated that SNPs in OAS-1 are associated with HCV recurrence within 12 months post-orthotopic liver transplantation (OLT) and OAS-1 SNP genotypes were significantly associated with the development of fibrosis within the first year. Additionally we observed an association between the SNP genotypes of OAS-1 and ISG-15 and CMV infection post-OLT. A significant distribution of ISG-15 genotypes was also found to correlate with acute rejection. These findings might help identify patients at high risk of developing complications within the first year.