Flavonoide sind weitverbreitete sekundäre Pflanzeninhaltsstoffe. Ihr Beitrag zur Prävention von chronischen Erkrankungen wird zu großen Teilen auf immunmodulatorische und neuroprotektive Effekte zurückgeführt. Eine Voraussetzung für die Nutzung dieser Eigenschaften der Flavonoide stellt die Erfassung cytotoxischer Effekte dar. Mit Ausnahme von Xanthohumol und Quercetin ist für alle im Rahmen der vorliegenden Arbeit untersuchten Flavonoide, Hispidulin, Baicalein, Scutellarein, Hesperetin, Chrysin, Apigenin, Naringenin, Catechin, Pelargonidinchlorid und EMD 21388, sowohl in T-Zellen (Jurkat) als auch in neuronalen (SK-N-SH)-Zellen nach 24-stündiger Inkubation eine geringgradige Cytotoxizität festzuhalten. Für Xanthohumol bzw. Quercetin wird ein halbmaximaler Verlust der Zellvitalität je nach Modell in Konzentrationen von 33-45 µM bzw. 118-208 µM erreicht. Der weiterführenden Charakterisierung (zVAD, DNA-Laddering) ist zu entnehmen, dass die zellulären Veränderungen substanzabhängig differieren und sowohl nekrotische Mechanismen (Xanthohumol) als auch apoptotische Vorgänge (Quercetin) einschließen. Eine erhöhte Lipidperoxidation im oberen Dosisbereich lässt darüber hinaus auf eine Beteiligung von oxidativem Stress an den von Xanthohumol-induzierten nekrotischen Prozessen schließen. Eine positive Einflussnahme auf die Zellvitalität durch Antioxidantien wie GSH und NAC lässt des Weiteren vermuten, dass die erfassten Flavonoid-induzierten Prozesse jeweils sensitiv zum Redoxzustand der Zelle sind. Während die Effekte von Xanthohumol auch in anderen Zellmodellen (HL-60) nachweisbar bleiben, verhält sich Quercetin nicht durchgehend vitalitätsmindernd. Unterschiede zwischen den Testsubstanzen bestehen auch hinsichtlich antioxidativer Effekte. Das Eliminieren freier Radikale zählt zu den wichtigsten Mechanismen, die bei Flavonoid-vermittelter Neuroprotektion eine Rolle spielen. Insgesamt sind alle diesbezüglich untersuchten Substanzen als starke Superoxidanionen-Radikalfänger einzustufen. Im Co-Inkubationsversuch zeigt Scutellarein den stärksten Effekt, gefolgt von Quercetin, Hispidulin und Xanthohumol. Im Prä-Inkubations-Versuchsmodell liegen in der Reihenfolge ihrer Effektstärken Xanthohumol vor Quercetin, Hispidulin und schließlich Scutellarein. Die modellabhängigen Konstanten können, unter Beteiligung einer passiven Diffusion der hydrophoben Flavonoidaglykone, auf eine substanzgebundene Membranpermeabilität zurückzuführen sein. Das antioxidative Potential der Flavonoide resultiert u.a. aus einer komplexen Einflußnahme auf die Genexpression in der Zelle. In der vorliegenden Arbeit sind anhand von cDNA-Arrays für mehrere Vertreter übereinstimmend Wechselwirkungen mit Genen der zellulären Abwehr dargestellt. Demnach führen Scutellarein, Hispidulin, Quercetin und Xanthohumol zu einer deutlich reduzierten Expressionsstärke von STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 und SOD2. Unter den Flavonoid-induzierten Veränderungen ragen die Effekte auf ADAR1 heraus, dessen Genexpression von Scutellarein bis auf ein 0,1-faches der Referenzwerte reduziert wird. Gleichsinnige Auswirkungen von Scutellarein auf die Expression von ADAR1-Protein in Western Blots unterstreichen diese Interaktion und legen nahe, dass ADAR-vermittelte enzymatische Deaminierungen durch Flavonoide moduliert werden können. Diese Beobachtung wird ergänzt durch den nachgewiesenen Effekt von Flavonoiden auf die Expression einer Reihe weiterer Gene (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F und APOBEC3G), die analoge posttranskriptionale Mechanismen steuern und gleichermaßen in Immunabwehr und Neuroprotektion eingebunden sind. Zu den wichtigsten Substraten von ADAR zählen Glutamatrezeptoren. Erwartungsgemäß ist nach der Einwirkung von Scutellarein auf humane Zellen, die Glutamatrezeptoren exprimieren, ein Rückgang der Deaminierung im Bereich der Glutamatrezeptoruntereinheit GluR 2 zu verzeichnen (Q/R-Position). Dem entspricht in elektrophysiologischen Modellen eine gesteigerte Ca2+-Permeabilität der jeweiligen Ionenkanäle und eine veränderte neuronale Exzitabilität. Hieraus ergibt sich ein breites Spektrum zusätzlicher Optionen für die Induktion von gesundheitsrelevanten Flavonoidfunktionen in der Zelle. So spielt die Modulation von Deaminierungen zugleich eine entscheidende Rolle im Vermehrungszyklus viraler Erreger. Die Annahme einer möglichen antiviralen Qualität von Scutellarein wird durch ein HBV-Infektionsmodell anhand drei Parameter der Virusreplikation (Virus-DNA-Konzentration, HBs- bzw. HBe-Antigenproduktion) bestätigt. Offen bleibt auch nach ausführlicher Prüfung, ob der deutliche antivirale Effekt als das Produkt von Flavonoid-induzierten Veränderungen der Deaminierungsraten oder als Folge eines Effekts auf die virale Polymerase zu interpretieren ist. Die hier dargestellten Wirkmechanismen leisten einen Beitrag zum Verständnis der Bedeutung von Flavonoiden für neue Anwendungen in Neuroprotektion und Immunabwehr. / Flavonoids are common secondary plant metabolites that confer numerous nutritional health effects. Their role in preventing chronic diseases is attributed to immunmodulatory and neuroprotective effects among others. In order to fully exploit these properties the limitations imposed by the compounds cytotoxic profiles must be addressed. For the majority of compounds investigated, hispidulin, baicalein, scutellarein, hesperetin, chrysin, apigenin, naringenin, catechin, pelargonidinchloride and EMD 21388, the present study confirms minimal cytotoxicity in T-cells (Jurkat) and in neuronal cells (SK-N-SH). As for xanthohumol and quercetin a 50% decline in cell-vitality is observed at concentrations of 33-45 µM and 118-208 µM, respectively. Further characterization using zVAD and DNA-laddering indicate that cell-vitality may be compromised both by necrotic mechanisms (xanthohumol) and by apoptotic effects (quercetin). An increase in lipidperoxidation in the upper dose range suggests that oxidative stress may be involved in xanthohumol toxicity. As this is counteracted by antioxidants such as GSH and NAC, these flavonoids impact on cell-vitality is likely codetermined by the cells redox state. While the effects of xanthohumol extend to other cell models, quercetin toxicity in HL-60 cells is less pronounced. Test compounds are also found to differ with regard to antioxidative profiles. The elimination of free radicals is a key mechanism in flavonoid-induced neuroprotection and is shown to vary with different incubation protocols. In short incubation experiments (5 min; co-incubation) scutellarein is identified as the most powerful scavenger, followed by quercetin, hispidulin and xanthohumol. In prolonged incubations (24 hrs; prä-incubation) xanthohumol and quercetin are followed by hispidulin and scutellarein. Model-specific constants suggest that passive diffusion of the hydrophobic flavonoid-aglyca may occur across cell membranes, alongside with other modes of permeation. Flavonoids antioxidative potential is mediated by complex effects on gene expression. The present work uses data from cDNA-arrays to highlight interactions with genes involved in cellular defense. Specifically, scutellarein, hispidulin, quercetin and xanthohumol downregulate expression for STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 and SOD2. In addition, flavonoids consistently downregulate ADAR1-expression, which drops to 0,1-fold of reference values and is paralleled by scutellarein-effects on ADAR1-protein-expression. Together, these findings indicate, that ADAR-mediated enzymatic deamination may be modulated by flavonoids. Similar effects are noted on related genes (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F and APOBEC3G), relevant to posttranscriptional processing underlying immune defense and neuroprotection. Glutamate receptors count among the most important neuronal substrates of ADAR. Following exposure to scutellarein a decrease in deamination rates is confirmed with respect to the glutamate receptor subunit GluR 2 (Q/R-site). As a result, an enhanced Ca2+-permeability of the respective ion channels is anticipated, and modified neuronal excitability. Overall, the regulation of enzymatic deamination by flavonoids offers opportunities for multilevel balancing of cell homeostasis. Thus deaminations may interfere with the replication cycle of viral pathogens. Using an ex-vivo HBV-infection model and three parameters of viral replication (viral load, HBs and HBe indices), antiviral properties of scutellarein are illustrated. Despite extensive investigation, it remains to be seen whether these effects can be ascribed to deaminations of viral DNA or to an interaction with other substrates, e.g. the viral polymerase. In summary, the present observations serve to foster our understanding of flavonoids roles in neuroprotection and immune defense.

Die Infektion mit dem Hepatitis C Virus (HCV) gilt als eine der Hauptursachen für chronische Hepatitiden und führt häufig zu Leberzirrhose und Leberkarzinom. Weltweit sind etwa 200 Millionen Menschen mit diesem Virus infiziert. Die aktuelle Behandlung der Hepatitis C mit Ribavirin und Interferon-alpha ist langwierig, beeinträchtigt durch Nebenwirkungen und führt nur bei einem Teil der Patienten zur Heilung. Aus diesem Grund ist die Entwicklung eines präventiv oder therapeutisch einsetzbaren Impfstoffes gegen HCV-Infektionen sehr wünschenswert. Das hoch attenuierte und in seiner Vermehrungsfähigkeit extrem eingeschränkte modifizierte Vakziniavirus Ankara (MVA) gehört zu den viel versprechendsten Kandidaten für die Entwicklung neuartiger rekombinanter Virusimpfstoffe. Im Rahmen dieser Arbeit sollten erste rekombinante MVA-HCV-Viren auf ihre Eignung als Impfstoffe untersucht werden. Als Zielantigene dienten wichtige virale Strukturproteine, darunter das unter den HCV-Genotypen hoch konservierte Nukleokapsidprotein Core, sowie das Nichtstrukturprotein NS3, welches als regulatorisches Virusprotein im HCV-Replikationszyklus eine wichtige Rolle spielt, untersucht werden. Hierfür wurden die rekombinanten MVA-Viren MVA-P7.5-HCV core (MVA-core) und MVA P7.5-HCV-1-830 (MVA-1-830) eingesetzt, welche für die HCV-Strukturproteine codierende Gensequenzen unter der Kontrolle des Vakziniavirus-spezifischen Promotors P7.5 exprimieren. Zusätzlich wurde ein weiteres rekombinantes Virus MVA-P7.5-HCV-NS3 (MVA-NS3) konstruiert, welches die Gensequenz für das HCV-Nichtstrukturprotein NS3 trägt. Alle Vektorviren erwiesen sich in in vitro Experimenten als genetisch stabil, erlaubten die Produktion der rekombinanten HCV-Antigene in infizierten Zielzellen und waren somit geeignet für in vivo Untersuchungen im Mausmodell. Da HCV-spezifischen CD8+-T-Zellantworten eine wichtige Rolle bei der Ausheilung einer Hepatitis C zugeschrieben wird, sollte insbesondere die Anregung dieser Immunantworten untersucht werden. Dabei zeigte sich, dass bereits eine einmalige Immunisierung mit MVA-core, MVA-1-830 oder MVA-NS3 ausreichend ist, um HCV-spezifische CD8+-T-Zellantworten zu induzieren. Diese CD8+-T-Lymphozyten konnten ex vivo in Epitop-spezifischer Weise zur Interferon-gamma-Synthese stimuliert werden, ließen sich Antigen-spezifisch in vitro expandieren und waren in der Lage, HCV-spezifische Zielzellen zu erkennen und zu lysieren. Zudem konnte eine Steigerung der Immunantworten durch Mehrfachapplikation der MVA-Vakzinen erzielt werden. Im Folgenden gelang es, die HCV-spezifischen CD8+-T-Zellantworten durch kombinierte Applikation der MVA-Vakzinen mit anderen rekombinanten Virusimpfstoffen wie Semliki-Forest-Viren oder Adenoviren, sowie mit Plasmid-DNA weiter zu verstärken. Solche Impfstrategien sind viel versprechend, da sich die gemeinsame Komponente der eingesetzten, unterschiedlichen Vektorvakzinen auf die rekombinanten Antigene beschränkt und eine starke Immunreaktion auf diese Antigene angeregt wird. Die in dieser Arbeit gewonnenen Erkenntnisse erlauben die Schlussfolgerung, dass rekombinante MVA-Vektoren, die HCV-spezifische Antigene produzieren, dafür geeignet sind, um nach Impfapplikation HCV-spezifische zelluläre Immunantworten zu induzieren. Die im Tiermodell erarbeiteten, optimierten Immunisierungsstrategien liefern eine erste Grundlage für weitere Immunisierungsexperimente in Primatenmodellen und zur Planung erster klinischer Studien im Menschen. / Infections with hepatitis C virus (HCV) are considered as one of the main causes for chronic hepatitis and often lead to liver cirrhosis and hepatocellular carcinoma. About 200 million people worldwide are chronically infected with this virus. The current antiviral therapy relying on ribavirin and interferon-alpha is time consuming, often impaired by side effects and leads to resolution of the disease in only a part of the patients. For this reason, the development of a prophylactic or therapeutic vaccine against HCV infections is very desirable. The highly attenuated and replication deficient modified vaccinia virus Ankara (MVA) is one of the most promising candidates for development of new generation virus vaccines. Purpose of this work was to evaluate first recombinant MVA HCV viruses for their suitability as vaccines against hepatitis C. HCV structural proteins, amongst them the highly conserved core protein, as well as the non-structural protein NS3, which plays a key regulatory role in the HCV replication cycle, served as target antigens for MVA vaccine development. First, we investigated recombinant MVA viruses MVA-P7.5-HCV-core (MVA-core) and MVA-P7.5-HCV-1-830 (MVA-1-830), which express the coding gene sequences for HCV structural proteins under control of the vaccinia virus specific promoter P7.5. Second, we constructed and characterized a recombinant virus MVA-P7.5-HCV-NS3 (MVA-NS3) that carries the gene sequence for the HCV non-structural protein NS3. As demonstrated by in vitro experiments, all vector viruses were genetically stable, permitted the production of recombinant HCV antigens in infected target cells and were thus suitable for in vivo experiments using mouse models. Since HCV specific CD8+ T cell responses are considered important in hepatitis C virus clearance, special emphasis was given to the analysis of induction of this kind of immune response. When tested in first vaccination experiments, already a single immunization with MVA-core, MVA-1-830 or MVA-NS3 was sufficient to induce HCV specific CD8+ T cell responses. These CD8+ T lymphocytes could be stimulated ex vivo in an epitope specific manner, resulting in interferon-gamma production, could be further expanded in vitro and were able to recognize and lyse HCV specific target cells. Additionally, multiple applications of the MVA vaccines resulted in an increase of these cellular immune responses. In a final series of experiments, the possibility to further amplify HCV specific CD8+ T cell responses could be demonstrated by using combined applications of MVA with other experimental gene transfer vaccines based on Semliki Forest virus, adenovirus or plasmid DNA. Overall, the results of this work clearly suggest that recombinant MVA vectors delivering HCV specific antigens, are suitable candidate vaccines for induction of HCV specific cellular immune responses upon immunization. Importantly, the definition of optimized immunization strategies offers a rational basis for further immunization studies in primate models and for the conception of first clinical studies in humans.

Ph.D. thesis, Faculty of Health Sciences, University of the Witwatersrand, 2009

Thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science (Biochemistry), University of the Witwatersrand, Johannesburg, 1992 / An investigation has been made into aspects of the expression of oncogenes in normally dividing cells and in hepatoceilular carcinoma (Hee). HOC occurs commonly in Southern Africs, and thf1aetiology ·ofthis tumour lsaseccieted with hepatitis a virus (HBV) infection. c·erbA, c..mva and e-tos but not c~Ha..res mANA were elevatad in tumours and adjacent hepatic tissue from the same petiEJ;htswhen compared to normal liver. Amounts of Fos and MYQ prot~in in the liver tumour specimens were else raised. The"e was some correlation between the patients' serum a..fetoproteirt concentretlons, histological features of tumour differentiatic)t"l, c..mvc and c40s r.ixpression. expression of e-tas and c..myc has been reportec to be elevated after stimulation of cells to alvlde, ,'1$ occurs during liver r19ganeration. This was corroborated by the findin~ that c-mvc, c·fo~· and c-jun mRNA concentratlona "Jere increased it"! cultured 3T6 mouse fibroblasts following treatment with alkaline medium aa a mitogenlo stimulus. The time course of the expression of these oncogenes was similar to that reported after gro\l'l/th factor sttmulation, The H[~V X..gene ma\' be responsible for increased oncogene expression it' YCC as a result of its documented trans activating properties. This vi!'a~ gene is unusual in that it has a codon preferanc";which is similar to that of eukarvotic ceU genes. Also HBV may ha'V& evolved from ti similar ancestral virus to that giving rise to retroviruses. These ideas suggest that the HBV X·gene is a viral oncogene derived from a host homologue. Low stringency Northern brot hybridisation using a X-gene probe denlonstrated a murine transcrlpt in heart and thymus. Attempts to isolate the sequence from mouse heart and thymus eDNA libraries ware unsuccessful despite ext,~n$jve screening with sensitive probes (SP6 palymerfjsa and peR fab(':.lUed X~gen~~fragments). Conserved X~gene \ . I sequences were also used fot the desigr:Jof primers in .~.peR bas£'d method " . II aimed at isolating a mammalian sequence. No sinnificant sequsnce \\ homology was found bet\lveen the HBVI\X..gene and Ol\A ampllfle'd from \1 l! gen(llmic and eDNA I1br'srytemplate sou~\pes.The peR preducts ttppeared to have been artef.,ots of arnplWaation. ~~n'IJreto detect the hQrtll.)logous gene may have resu~ted from poo' complS,JIlentarity between the VIral ant! \\ mammalian secuencec, 1\ \\ Non..~pecific amplification is commonly enct~unter&d when u$1110 PCli'. A qtJick asvmmatrlc re·ampW~catj(ii1 method I,?ssed on eXUOSilin of an " interm.uly' hybrfdising X·gelllapfimar we! davisQ\j to confirm FICRprOdu(,ts. The l"n1ithodwas specific irlthat "ver~ single bas~ mlsmatohe$ betwsen the internal primer and tem1>late re;.,ultad in fatJut~ of dete(;tabla \tUim$f extension. / GR 2016

Master of Science / The hepatitis B virus (HBV) vaccine has contributed greatly to decreasing the HBV epidemic. However, it remains unclear why 5-10% of individuals do not mount an adequate antibody response. Previous studies have shown that genetic variation influences HBV vaccine response. Since such studies are lacking in South African individuals, we examined the associations between HBV vaccine response and genetic variation in HLA-DPB1, additional candidate genes and HLA-DPB1 expression levels in a South African cohort. HLA-DPA1 and -DPB1 allele typing was performed using Luminex technology, twenty-four candidate SNPs were typed by MassArray Analysis and HLA-DPB1 mRNA expression levels were measured by qPCR. HLA-DPB1*01:01, *04:01:01G and *09:01 and SNPs and haplotypes in IL1B, IL4, IL12B, IFNG and the HLA region were significantly associated with HBV vaccine response. A trend of lower HLA-DPB1 expression associating with better anti-HBs response was observed, although this was not significant. Response to the HBV vaccine is multi-genic but HLA-DP plays an important role. / CR2017

Introdução: A estimativa de sobrevida de pacientes com HIV/aids aumentou após a terapia antirretroviral de alta potência: no entanto, a mortalidade por doenças hepáticas também cresceu. Objetivos: Estimar a probabilidade acumulada de sobrevida após o diagnóstico de aids entre pacientes coinfectados HIV/HCV e realizar análise exploratória para investigar fatores relacionados à sobrevida desses pacientes. Metodologia: Estudo de coorte não concorrente, utilizando sistemas de Informações: o de Agravos de Notificação, o de informação laboratorial e o de informação da vigilância epidemiológica do Centro de Referência e Treinamento DST/AIDS-SP, de pacientes com aids maiores de 13 anos, acompanhados no ambulatório geral. As variáveis estudadas foram: hepatite C, hepatite B, categoria de exposição, contagem de células T CD4+, faixa etária, escolaridade, cor, sexo e períodos de diagnóstico de aids: 1986 a 1993, 1994 a 1996, 1997 a 2002 e 2003 a 2010. Foi utilizado o estimador de Kaplan-Meier, o modelo de Cox e as estimativas das hazard ratio (HR) com os respectivos intervalos de confiança (IC 95 por cento ). Resultados: De um total de 2.864 pessoas incluídas, com idade mediana de 35 anos, 219 foram a óbito (7,5 por cento ). De 358 (12,5 por cento ) coinfectados, 159 (45,1 por cento ) eram usuários de drogas injetáveis (UDI) e de 2.506 não coinfectados, 96 (3,9 por cento ) eram UDI. A probabilidade acumulada de sobrevida entre coinfectados, a partir do diagnóstico de aids, foi 100 por cento aos 60 meses no período de 1986 a 1993; 27,8 por cento aos 168 meses no período de 1994 a 1996; 76,3 por cento aos 168 meses no período de 1997 a 2002 e 92,8 por cento aos 96 meses no período de 2003 a 2010. As curvas de sobrevida foram diferentes entre coinfectados e não coinfectados no período de 1994 a 1996 (log rank = 19,8; p < 0,001) e no período de 1997 a 2002 (log rank = 38,8; p < 0,001). No modelo de Cox multivariado, mostraram-se preditores de óbito, independentemente das outras variáveis: ter hepatite C (HR = 2,9; IC 2,1-3,9), ter hepatite B (HR = 2,5; IC 1,7-3,6), ter até 3 anos de estudo (HR = 2,3; IC 1,5-3,6), ter 50 anos ou mais de idade (HR = 2,1; IC 1,3-3,2). Ter diagnóstico de aids no período entre 1997 a 2002 mostrou-se fator de proteção ao óbito (HR = 0,4; IC 0,3-0,5). Conclusões: Coinfectados HIV/HCV apresentaram menor sobrevida quando comparado com não coinfectados nos períodos de diagnóstico de aids 1994 a 1996 e 1997 a 2002. A partir do período 1994 a 1996, observou-se aumento significativo na probabilidade acumulada de sobrevida entre coinfectados, sendo que no período 2003 a 2010, essa probabilidade foi semelhante entre coinfectados e não coinfectados, refletindo possível impacto do tratamento da hepatite C / Introduction: The estimated survival of patients with HIV/AIDS has increased after highly active antiretroviral therapy; mortality due to liver diseases, however, has also increased. Objectives: To estimate the accumulated probability of survival after AIDS diagnosis among HIV/HCV co-infected individuals and to perform an exploratory analysis to investigate factors related to the survival of these patients. Method: Non-concurrent cohort study, using data from the National Disease Reporting Information System, the laboratory and epidemiological surveillance information systems of the SP-STD Reference and Training Center-CRT, of patients over 13 years of age, followed at the general outpatient clinic. The following variables were studied: hepatitis C, hepatitis B, exposure category, T CD4+ cell count, age group, schooling, color, sex, and AIDS diagnostic periods: 1986 to 1993, 1994 to 1996, 1997 to 2002 and 2003 to 2010. Survival analysis was performed using the Kaplan-Meier estimator and the Cox model, with estimates of the hazard ratio (HR) and respective confidence intervals (95 per cent CI). Results: Of a total of 2,864 individuals included, with a median age of 35 years, 219 died (7.5 per cent ). Of the 358 (12.5 per cent ) HIV/HCV co-infected individuals, 159 (45.1 per cent ) were injecting drug users (IDU), and of the non-co-infected 2,506, 96 (3.9 per cent ) were IDU. The accumulated probability of survival among HIV/HCV co-infected individuals at 60, 168, 168 and 96 months as of AIDS diagnosis, was 100 per cent in the 1986 -1993 period; 27,8 per cent in the 1994-1996 period; 76,3 per cent in the 1997-2002 period; and 92,8 per cent in the 2003-2010 period. The survival curves were different between co-infected and non-co-infected individuals in the 1994-1996 (log rank = 19,8; p < 0,001) and in the 1997-2002 (log rank = 38,8; p < 0,001). In the multivariate model, regardless of other variables, the following were predictors of death: having hepatitis C (HR = 2.9; CI 2.1-3.9); having hepatitis B (HR = 2.5; CI 1.7-3.6); being 50 years old or over (HR = 2.1; CI 1.3-3.2) and having up to 3 years of schooling (HR = 2.3; CI 1.5-3.6). AIDS diagnosis between 1997 and 2010 was shown to be a protective factor for death (HR = 0.4; CI 0.3-0.5). Conclusions: HIV/HCV co-infected individuals had shorter survival, when compared to non-co-infected individuals in the 1994-1996 and in the 1997-2002 AIDS diagnostic periods. As of the 1994-1996 period, a significant increase in the accumulated probability of survival among HIV/HCV co-infected individuals was observed. In the 2003-2010 period, the probability was similar between co-infected and non-coinfected individuals, showing the possible impact of hepatitis treatment

Foram estudados 151 doadores de sangue encaminhados pelo Hemocentro de Ribeirão Preto ao Ambulatório de Hepatites do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto em função de terem apresentado resultados foram positivos para hepatite C nos testes de triagem pré-doação, entre 2001 e 2010. Todos tiveram confirmação diagnóstica mediante uso de técnicas de biologia molecular. No momento de chegada ao Hospital foram entrevistados por uma assistente social ligada ao Núcleo Hospitalar de Epidemiologia, com o objetivo de caracterizá-los segundo variáveis sociodemográficas, estudar fatores de risco presentes e genótipos encontrados. Houve predominância de indivíduos do sexo masculino, com baixos níveis de escolaridade e de estratos sociais menos favorecidos, com idade mediana de 36 anos. Observou-se uma tendência decrescente de infectados ao longo dos anos estudados. O genótipo mais prevalente foi o 1 com percentual de 68,8%, seguido do genótipo 3 (27,0%). Os potenciais fatores de risco mais prevalentes foram história pregressa de hospitalização sem e com cirurgia, multiplicidade de parceiros sexuais no passado, convivência com usuários de drogas, contato com sangue em atendimentos a terceiros, múltiplos parceiros sexuais no presente e contato domiciliar com casos de hepatite. Também com elevadas frequências foram observados os seguintes fatores: antecedente de transfusão sanguínea, uso coletivo de escova de dentes, história de contato sexual com usuários de drogas, frequência a creches na infância e tatuagem. Uso passado de drogas injetáveis e compartilhamento de seringas e agulhas foram relatados por 15,2% e 9,9%, respectivamente. Conclui-se que deve ter ocorrido omissão de inúmeros fatores de risco por ocasião da triagem clínica, possivelmente indicativo do desejo de se utilizar o Banco de Sangue como controle da situação sorológica por parte de parte dos doadores. / 151 blood donators were analyzed sent from the Hemocenter of Ribeirão Preto to the Hepatitis Clinic of the University Hospital of the Ribeirão Preto Medical School, University of São Paulo, because these individuals had presented positive results for Hepatitis C in the screening tests for pre-donation, taken between 2001 and 2010. All of them had diagnosis confirmation through the use of molecular biology techniques. At the moment of arrival in the hospital, they were interviewed by a Social Assistant from the Hospital Nucleus of Epidemiology, in order to characterize them according to socio-demographic variables, to study risk factors that might be present, and genotypes found. The predominance was of male individuals, with low school levels, and coming from a less favored social stratum, with a median age of 36 years. There was a decreasing tendency of infected individuals along the years of study. The most prevailing genotype was type 1 with a percentage of 68.8%, followed by the genotype 3 (27.0%). The most prevailing potential risk factors were previous history of hospitalization with and without surgery, multiplicity of sexual partners in the past, acquaintance with drug user, contact with blood on dealing with to third parties, multiple current sexual partners and home contact with hepatitis cases. The following factors were also observed with high frequency: antecedent of blood transfusion, collective use of tooth brush, history of sexual contact with drug users, attendance to day care clinics in childhood and tattoos. Past use of injectable drugs and syringe and needle sharing were reported by 15.2% and 9.9%, respectively. The conclusion was that there must have been the omission of a number of risk factors at the time of the clinical screening, possibly indicating the desire of using the Blood Bank to make control of the serological status by some of the donators

Orientador: Hideko Yamanaka / Banca: Assis Vicente Benedetti / Banca: Antonio Aparecido Pupim Ferreira / Banca: Maria Isabel Pividori Gurgo / Banca: Neiva Sellan Lopes Gonçales / Resumo: No presente trabalho estudou-se a construção de um genossensor e sua viabilidade para compor um dispositivo de análise rápida que pudesse ser aplicado na realização do diagnóstico e genotipagem do vírus da Hepatite C (HCV). Primeiramente otimizou-se uma metodologia para a construção dos eletrodos de carbono impresso, os quais foram empregados como dispositivo transdutor. O genossensor foi, então, construído pela imobilização da sonda de captura na superfície do eletrodo de carbono. A sua superfície foi eletroquimicamente oxidada para a geração de grupos carboxílicos, os quais foram utilizados para a imobilização da proteína estreptavidina (STA). A sonda de captura biotinilada foi, então, imobilizada via interação biotina-STA, sendo os sítios remanescentes da STA bloqueados com biotina. Monitorou-se o evento biológico de hibridização entre a sonda de captura e sua sequência complementar via corrente de oxidação da base nitrogenada guanina. Para tanto foi empregada a técnica de voltametria de onda quadrada. Este genossensor demonstrou alta seletividade frente às sequências de oligonucleotídeos contendo uma e quatro bases não complementares e sequências provenientes de viroses coinfectantes do HCV (HIV e HBV). O genossensor estudado foi aplicado na identificação de amostras provenientes de pacientes HCV positivos (HCV do tipo 1 e 3) e negativos. O estudo das amostras negativas possibilitou a determinação de um valor de cut-off de 37 μA, o qual foi utilizado na genotipagem das amostras classificadas como positivas. Em seguida, construiu-se uma célula de detecção com volume de 25 μL utilizando a cerâmica LTCC (do inglês, Low Temperature Co-Fired Ceramic). Os eletrodos de trabalho, referência e auxiliar foram confeccionados dentro da célula utilizando tintas condutoras... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the present work was studied the construction of a genosensor and its availability to compose a device for fast analysis which could be applied in Hepatitis C virus (HCV) diagnose and genotyping. Firstly the methodology for screen printed electrodes' construction was optimized and these electrodes were used as transducer device. Then the genosensor was constructed immobilizing the capture probe on the electrode surface. Thereunto the surface was electrochemically oxidized to carboxylic groups formation, which were used to streptavidin (STA) immobilization. After the biotinilated probe was immobilized through biotin-STA interaction, and the STA reminiscent sites were blocked with biotin solution. Hybridization between the capture probe and its complementary sequence was monitored by means of the guanine oxidation current. Square wave voltammetry was used to this end. The final genosensor showed high selectivity when incubated in solution containing sequences with one and four non complementary nitrogenous bases and also with sequences of coinfecting viruses (HIV e HBV). This genosensor was applied in the identification of samples of HCV positives (HCV type 1 and 3) and negatives patients. This study allowed the determination of the cut-off value (37 μA) which was used in the HCV positive genotyping process. Then a 25 μL detection cell was constructed using LTCC ceramic. The working, reference and auxiliary electrodes were prepared inside the cell by means of conducting inks. The cell electrochemical behavior was evaluated in a standard system (with the redox pair Fe(CN)6 3+/Fe(CN)6 4+). Finally, the genosensor was constructed inside the detection cell using carbon nanotubes as amplifier of the guanine oxidation signal. A very good performance to the conjoint was observed, which means that this... (Complete abstract click electronic access below) / Doutor

Infection with hepatitis C virus (HCV) can cause liver damage known as fibrosis, which often leads to liver disease and hepatocellular carcinoma. The impairment of circulating, bulk (non-specific and specific) CD8+ T cells within HCV-infection, characterized by an altered phenotype and the increased expression of pro-apoptotic genes, is observed when compared to uninfected controls. The relationship between bulk CD8+ T cell function and the extent of liver damage has not been demonstrated. In this study, widespread immune alterations were observed in untreated HCV infection with advanced liver fibrosis. Untreated HCV-infected individuals with advanced fibrosis possessed a significantly decreased proportion of naïve CD8+ T cells and an increased proportion of late effector memory CD8+ T cells compared to uninfected controls. Upon T cell receptor (TCR) stimulation, these individuals also had an increased intracellular IFN-γ expression for four CD8+ T cell subsets, a decreased CD107a expression for central memory CD8+ T cells, and a decreased perforin induction for naïve and central memory CD8+ T cells. These immune alterations did not reverse 24 weeks after viral cure. This study indicates there is a relationship between the differentiation and function of bulk CD8+ T cells and the extent of liver damage within HCV infection.

Recentemente estudos demonstraram que os SNPs (polimorfismos de base única) rs8099917 e rs12979860 localizados próximos ao gene da IL28B explicam a variação de resposta à infecção e tratamento do paciente contra o genótipo 1 do HCV, porém não para o genótipo 3 deste vírus. Este trabalho encontrou associação significativa entre resposta à infecção devida ao genótipo 3 pelo tratamento (PEG-INF e RBV) e o polimorfismo rs8099917 em uma amostra da população de Santos - SP. Para o polimorfismo rs12979860, esta associação somente foi encontrada ao se parear indivíduos para sexo, idade e grau de fibrose hepática, demonstrando a importância da retirada de efeitos de estratificação neste tipo de análise. Estes resultados se confirmam ao se agregar dados de uma população proveniente da Bahia em uma meta-análise. Além disso, fez-se um estudo GWAS a fim de se conhecer outras variações genéticas envolvidas nessa resposta. Esta análise indicou a existência de alguns SNPs candidatos com sugestão de associação, dentre eles a tiroglobulina, relacionada aos hormônios da tireóide. / Recently, studies have shown that SNPs (single nucleotide polymorphisms) rs8099917 and rs12979860, located near the gene IL28B explain the changes in the response to infection and treatment of a patient against the HCV genotype 1, but not for the genotype 3 of the virus. This study found a significant association between response to infection due to treatment by genotype 3 (PEG-INF and RBV) and the rs8099917 polymorphism in a population sample from Santos - SP. To the rs12979860 polymorphism, this association was only found when individuals are paired for sex, age and degree of hepatic fibrosis, demonstrating the importance of the withdrawal effects of stratification in this type of analysis. These results confirm the aggregate data from a population of Bahia in a meta-analysis. In addition, a GWAS was made in order to search other genetic variations involved in this response. This analysis indicated the existence of some candidate SNPs with suggestion of association, including thyroglobulin, thyroid hormones related to.