This study involved the analysis of 112 patients with autoimmune hepatitis (AIH), during which process several specific issues or problems were identified. One subgroup included pregnant patients, and another patients diagnosed at an older age (>65 years). The diagnosis of AIH is not always easy, and the investigations revealed that some patients diagnosed with AIH had features of an overlap syndrome (with primary sclerosing cholangitis) or in fact never had AIH, but as retrospectively diagnosed, had toxic hepatitis masquerading as AIH. Some patients who were initially documented as clearly having chronic hepatitis C, also had autoantibodies in their serum and were (or could have been) incorrectly diagnosed as AIH. The results showed a large variability in symptoms and complaints at the time of diagnosis of AIH. The disorder can present as an acute or chronic disease. In some patients only vague symptoms are reported and in others, the diagnosis is an incidental discovery because of abnormal liver enzymes. In contrast, other patients present with acute liver failure. The diagnosis must be considered in patients with non-viral liver disease with significantly raised ANA and SMA and high serum gammaglobulin concentrations. No fixed age predisposition was found, but rather an even spread over various age groups. The male:female ratio is 1:2.5. This disorder can for the most part be well treated with a combination therapy of corticosteroids and azathioprine. Experience shows that high doses of corticosteroids can cause serious complications, particularly in older patients. A lower dose is mostly adequate, although biochemical normalisation may take longer. In approximately half of the patients, corticosteroid-therapy was discontinued, with azathioprine as the only treatment. The long-term survival of adequately treated patients is almost equal to that of the control group. In the cohort, ten patients died: three who had presented with acute liver failure, two who later developed a hepatocellular carcinoma and one who died because of cerebral lymphoma. Three older patients died of sepsis, possibly co-induced by high doses of steroids. Five patients underwent liver transplantation, of which one died of aspergillosis. / Dissertation (MD)--University of Pretoria, 2011. / Internal Medicine / unrestricted

The purpose of this study was to identify mutations in the basic core promoter and enhancer II region a* the hepatitis B virus (HBV) that might result in the hepatitis B virus e antigen (HBeAg)-negative phenotype and contribute to hepatocarcinogenesis in black African carriers of the virus. The basic core promoter/enhancer II overlaps the X gene. HBV DNAfrom serum of 47 asymptomatic carriers and 50 patients with hepatocellular carcinoma and from 29 tumorous and 10 nontumorous liver tissues was amplified and sequenced directly. That part of the enhancer II region not overlapping the basic core promoter was reasonably well conserved in all samples. Missense mutations at positions 1809 and 1812 were found in 80% of all sequences and may represent wiidtype sequence in southern African isolates. Nucleotide and amino acid divergences were higher in the basic core promoter of hepatocellular carcinoma patients than of asymptomatic carriers (p<0.0001). This applied particularly to the paired 1762 adenine to thymine (1762T) and 1764 guanine to adenine (1764*) missense mutations, the prevalence of which was 66% in patients with hepatocellular carcinoma compared with 11% in asymptomatic carriers (p<0.0001). There was no association between the presence of 1762T1764A and the rate of HBeAg negativity, although these mutations suppressed HBeAg titres in HBeAg-positive patients. Suppression of HBeAg expression as well as alteration of amino acid sequence of the X protein may be contributing factors in the development of hepatocarcinogenesis.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2017 / Hepatitis B is a global health problem that kills about 600 000 people annually. It is an infectious disease caused by the Hepatitis B Virus (HBV), which infects the liver and leads to liver inflammation and secondary complications such as cirrhosis and Hepatocellular Carcinoma (HCC). The available therapies are only partially effective and are associated with adverse side effects and viral drug resistance. RNA interference (RNAi) pathway is a gene silencing pathway found in diverse living systems including mammals. Harnessing of this pathway to inhibit HBV replication has shown a lot of promise, with highly effective anti-HBV RNAi activator sequences designed. However, the lack of safe and efficient delivery and expression systems for these sequences is one of the obstacles that need to be overcome before RNAi effectors can reach clinical application. For easy assessment of transduction efficiency, Helper Dependent Adenoviral vectors (HDAd) expressing β-galactosidase (encoded by lac Z gene) are commonly used to deliver anti-viral RNAi activators. However, this reporter protein has been blamed for induction of innate immune response and concomitant clearance of the HDAds by the host. For the first time, this study explored the use of lac Z-deficient HDAds to deliver anti-HBV RNAi activators expressed under the control of a liver-specific phosphoenolpyruvate carboxykinase (PEPCK) promoter. HDAd expressing Firefly luciferase resulted in a significant luminescence detected in cell culture lysates and a sustained bioluminescence in mice. HDAds expressing anti-HBV sequences transduced the liver efficiently and did not induce a pronounced inflammatory response or liver toxicity in mice. However, this did not translate into maximal anti-HBV sequence expression and HBV replication inhibition in vitro and in vivo. This suggests that the PEPCK promoter is inadequate for RNAi activator expression. This study highlights the importance of careful RNAi activator regulatory elements selection and presents the therapeutic potential and utility of HDAd vectors for hepatotropic delivery of antiviral sequences with markedly attenuated immune response stimulation and toxicity. For improved anti-HBV RNAi activator expression and HBV knockdown, a different liver specific promoter mouse transthyretin receptor (MTTR) promoter is currently being investigated in our lab. / MT2017

No description available.

The hepatitis B virus (HBV), chronically infecting ~360 million people worldwide, accounts for over half of the cases of hepatocellular carcinoma (HCC) and contributes to over 650,000 deaths per year making HCC is the fifth most common and second most deadly form of cancer.1-3 Many genes deregulated by the hepatitis B x-antigen (HBx), the oncogenic protein encoded by HBV, are known drivers of HCC.4,5 The timing of these alterations in the initiation and progression of disease, however, are poorly understood and the treatment options for HCC are extremely limited. Here, RNA-Seq expression data originally from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project were used to compare truly healthy liver tissues, a base-line level often excluded from cancer studies, to both non-tumor and tumor tissues of patients with HBV associated HCC. This illuminated inflammation and immune response process as dysregulated prior to tumor formation followed by disruption of cell cycle and cell survival processes once tumors have arisen. Connecting these processes are UBD, BCL6, METTL24, CHRNA4, and NFKBIZ which putatively serve as crucial early drivers in the progression from HBV infection to the development of HCC and affect the serum level of downstream targets which could serve as biomarkers for earlier disease detection. Differential methylation analysis was also carried out on samples directly from TCGA and the Gene Expression Omnibus (GEO) to determine if the differentially expressed genes were potentially deregulated due to reversible epigenetic alterations. Enriched pathways for differential methylation in non-tumor samples included the immune system and the cell cycle but none of the genes of interest from differential expression analysis were differentially methylated until stage 1 indicating that methylation is involved in the progression of disease and not initiation. / Biology

Administration of Concanavalin-A (Con A) induces severe injury to the hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A induced hepatitis. Although immune cells have been identified as the causative agent of Con A-induced hepatitis, the exact mechanism of pathogenesis remains unclear. When Con A was injected into CD44 wild type (WT) mice, it induced hepatitis as evident from increased plasma aspartate aminotransferase (AST) levels accompanied by active infiltration of mononuclear cells in the liver and significant induction of apoptosis. Interestingly, Con A injected C57BL/6 CD44-knockout (KO) mice exhibited increased hepatitis with higher levels of apoptosis in the liver and increased plasma AST levels when compared to the CD44 WT mice. Also, transfer of T cells from Con A injected CD44-KO mice into CD44 WT mice induced higher levels of hepatitis when compared to transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44-KO mice was partially due to increased production of cytokines such as TNF-a, IL-2 and IFN-g, but not Fas or FasL. Also, it was not caused by altered presence of T cell subsets. The increased susceptibility of CD44 KO mice to hepatitis correlated with increased resistance of T cells from CD44 KO mice to undergo apoptosis when compared to the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis. / Master of Science

Zhu, Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-118). / Abstracts in English and Chinese. / Contents --- p.I / List of Abbreviations --- p.IV / List of Tables and Figures --- p.V / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Viral Hepatitis --- p.2 / Chapter 1.2 --- Global Epidemiology of HBV --- p.3 / Chapter 1.3 --- Modes of Transmission --- p.4 / Chapter 1.4 --- Diagnostic Tests --- p.5 / Chapter 1.4.1 --- HBeAg and Anti-HBe --- p.7 / Chapter 1.4.2 --- Serum Enzymes --- p.8 / Chapter 1.4.3 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.1 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.2 --- Clinical Applications of DNA Assays --- p.10 / Chapter 1.4.4 --- Histology --- p.13 / Chapter 1.5 --- Natural Course of Chronic Hepatitis infection --- p.18 / Chapter 1.5.1 --- Phases of chronic hepatitis B --- p.18 / Chapter 1.5.2 --- HBeAg-negative chronic hepatitis B --- p.21 / Chapter 1.6 --- Molecular biology of HBV --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Genomic structure and organization --- p.24 / Chapter 1.6.2.1 --- Surface ORF --- p.24 / Chapter 1.6.2.2 --- Precore/Core ORF --- p.25 / Chapter 1.6.2.3 --- Polymerase ORF --- p.25 / Chapter 1.6.2.4 --- X ORF --- p.26 / Chapter 1.7 --- Genetic Variation of HBV --- p.31 / Chapter 1.7.1 --- HBV genotypes --- p.31 / Chapter 1.7.2 --- Predominant genotypes and their subgroups in Asia --- p.33 / Chapter 1.7.3 --- HBV mutations --- p.36 / Chapter 1.7.3.1 --- Precore mutations --- p.37 / Chapter 1.7.3.2 --- Core promoter mutations --- p.38 / Chapter 1.7.3.3 --- Other Mutations associated with clinical outcome --- p.40 / Chapter Chapter Two: --- Methodology --- p.44 / Chapter 2.1 --- Aims and Hypothesis --- p.45 / Chapter 2.1.1 --- Aims --- p.46 / Chapter 2.1.2 --- Hypothesis --- p.47 / Chapter 2.2 --- Patient Recruitment --- p.48 / Chapter 2.3 --- Laboratory Assays --- p.49 / Chapter 2.3.1 --- Preparation of serum HBV DNA --- p.49 / Chapter 2.3.2 --- Quantification of serum HBV DNA --- p.51 / Chapter 2.4 --- Full-genome Amplification of HBV DNA --- p.53 / Chapter 2.5 --- Full-genome Sequencing of HBV DNA --- p.55 / Chapter 2.6 --- Assembly of HBV Full-genome Sequence --- p.58 / Chapter 2.7 --- Phylogenetic Analysis --- p.59 / Chapter 2.7.1 --- Construction of phylogenetic tree --- p.59 / Chapter 2.7.2 --- Genotype and subgenotype determination --- p.60 / Chapter 2.8 --- HBV Mutations --- p.62 / Chapter 2.9 --- Info-gain program --- p.64 / Chapter 2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Results --- p.67 / Chapter 3.1 --- Patient Information --- p.68 / Chapter 3.2 --- Phylogenetic Analysis --- p.69 / Chapter 3.3 --- HBV genotypes/subgenotypes --- p.76 / Chapter 3.4 --- “Hot-spo´tح HBV Mutants --- p.79 / Chapter 3.5 --- HBV Mutation Associated with Liver Fibrosis --- p.82 / Chapter 3.5.1 --- Mutant selection --- p.82 / Chapter 3.5.2 --- Clinical significance of novel mutants --- p.84 / Chapter Chapter Four: --- Discussion --- p.88 / Chapter 4.1 --- Full-genome Sequencing Strategy --- p.89 / Chapter 4.2 --- HBV genotypes/subgenotypes Distribution and Disease Activity --- p.90 / Chapter 4.2.1 --- HBV genotypes/subgenotypes distribution --- p.90 / Chapter 4.2.2 --- Clinical significance of genotypes/subgenotypes --- p.91 / Chapter 4.3 --- HBV Hotspot Mutants and Disease Activity --- p.93 / Chapter 4.4 --- HBV Novel Mutants --- p.96 / Chapter 4.5 --- Limitation of the Study and Future Work --- p.97 / Chapter 4.5.1 --- Limitation --- p.97 / Chapter 4.5.2 --- Future Direction --- p.98 / Chapter Chapter Five: --- Conclusions --- p.99 / References --- p.102

Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Since the advent of recombinant DNA technology in the 1970s, various microbial hosts have been employed for the efficient high-level heterologous production of a variety of proteins, ranging from enzymes and reagents to therapeutics and vaccines. More recent microbial hosts to be employed for these purposes are filamentous fungi, and particularly the genus Aspergillus. Aspergilli have been associated with industrial processes for many years and are used in the production of antibiotics, enzymes, citric acid and Oriental foods and beverages, and thus strains such as Aspergillus niger and Aspergillus oryzae have been afforded GRAS (Generally Regarded 8s ~afe) status. They also secrete copious amounts of homologous and heterologous proteins and are able to perform post-translational modifications effectively. Various proteins of pharmaceutical interest have been successfully expressed in Aspergillus, but the potential of these fungi to produce heterologous viral proteins has not been explored extensively. In this study, we evaluated the potential of the filamentous fungi A. niger and Aspergillus awamori as alternative hosts for the heterologous production of hepatitis B viral proteins. Hepatitis B is a serious, potentially lethal liver disease that affects 2000 million people world-wide and has a high endemicity in Southern Africa. Currently there is no effective treatment for viral hepatitis and thus a mass vaccination strategy is the only solution to curb the spread of the disease. This kind of vaccination strategy requires a cheap, safe and effective vaccine and these objectives have been achieved in the development of recombinant subunit vaccines from yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris that are commercially available. The hepatitis B virus envelope consists of a membrane fraction and three proteins, namely the major (S) protein (encoded by the S gene), the middle (M) protein (encoded by the preS2S gene) and the large (L) protein (encoded by the preS1preS2S gene). When produced in the above-mentioned yeasts, the S protein was shown to spontaneously assemble into pseudoviral particles devoid of viral DNA, which were then purified and used as vaccine. In the present study the Sand preS1preS2S genes from a local isolate of hepatitis B subtype adw2 were placed under transcriptional control of the constitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter and the inducible A. niger glucoamylase (glaA) promoter. The respective viral genes were also fused to the region encoding the catalytic domain of the highly expressed and secreted A. niger glucoamylase, which served as a carrier moiety to possibly facilitate viral protein secretion. The various gene constructs were subsequently transformed to laboratory strains of A. niger and A. awamori and numerous transformants were obtained. One A. niger transformant carrying the S gene under control of the gpdA promoter contained approximately 7 integrated copies of the expression cassette and produced hepatitis B pseudoviral particles intracellularly at levels of 0.4 mg/I culture. These levels are approximately ten-fold higher than those initially obtained from the yeast S.cerevisiae, which showed yields of 0.01 to 0.025 mg/I. None of the other transformants could be shown to produce recombinant S or L protein and no secretion of viral protein could be demonstrated. This could be attributed to numerous factors, including vector copy number, site of integration or proteolytic activity. The most important insight emerging from this work regarding secretion of heterologous viral protein was that the addition of a carrier protein hampered, rather than enhanced secretion of the viral envelope protein, due to the inherent properties of viral protein assembly. This work also serves as a "proof of principle", showing that Aspergillus is indeed a viable alternative host for the production of hepatitis B pseudoviral particles, and could be investigated further for its potential as host for the heterologous expression of other viral proteins. / AFRIKAANSE OPSOMMING: Sedert die ontwikkeling van rekombinante DNA tegnologie in die sewentigerjare is verskeie mikroorganismes reeds gebruik vir die doeltreffende produksie van 'n verskeidenheid proteïne teen hoë vlakke; onder andere ensieme, reagense, terapeutiese middels en vaksiene. Onlangs is filamentagtige swamme, veral van die genus Aspergillus, ontwikkel vir heteroloë proteïenproduksie. Aspergilli word al vir baie jare in nywerheidsprosesse gebruik, onder andere in die vervaardiging van antibiotika, ensieme, sitroensuur en sekere Oosterse voedsel- en drankprodukte. As gevolg van hierdie jarelange gebruik van rasse soos Aspergillus niger en Aspergillus oryzae, word hulle algemeen aanvaar as veilig vir menslike gebruik. Hierdie swamme besit veral die vermoë om hoë vlakke van homoloë en heteroloë proteïene uit te skei en die na-translasiemodifisering van proteïene korrek uit te voer. Verskeie proteïene van farmaseutiese belang is al suksesvol in Aspergillus uitgedruk, maar die potensiaal van hierdie swamme om virale proteïene te vervaardig is nog nie deeglik ondersoek nie. Hierdie studie ondersoek die geskiktheid van die filamentagtige swamme A. niger en Aspergillus awamori om as alternatiewe gashere vir die heteroloë produksie van hepatitis B proteïene te dien. Hepatitis B is 'n ernstige en selfs dodelike lewersiekte. Omtrent 2000 miljoen mense wêreld-wyd is met die virus geïnfekteer en dit is veral endemies in Suiderlike Afrika. Daar is tans geen doeltreffende behandeling vir virale hepatitis en dus is wêreld-wye inentingsprogramme die enigste oplossing om die verspreiding van die siekte te bekamp. Hierdie inentingsstrategie vereis die beskikbaarheid van 'n bekostigbare, veilige en doeltreffende vaksien. Die rekombinante subeenheidvaksiene wat ontwikkel is deur van gashere soos Saccharomyces cerevisiae, Hansenula polymorpha en Pichia pastoris gebruik te maak, voldoen aan hierdie vereistes en is kommersieel beskikbaar. Die omhulsel van die hepatitis B virus bestaan uit 'n membraangedeelte en drie proteïene, naamlik die hoofproteïen (S) (gekodeer deur die S-geen), die middelproteïen (M) (gekodeer deur die preS2S-geen) en die grootproteïen (L) (gekodeer deur die preS1preS2S-geen). Wanneer die S-proteïen in bo-genoemde giste uitgedruk word, vorm dit spontaan pseudovirale partikels wat nie virale DNA bevat nie. Hierdie partikels word dan gesuiwer en as vaksien gebruik. In hierdie studie is die S- en preS1preS2S-gene, vanaf 'n plaaslike isolaat van hepatitis B subtipe adw2, onder transkripsionele beheer van die konstitutiewe Aspergillus nidulans gliseraldehied-3-fosfaat-dehidrogenasepromoter (gpdA) en die induseerbare A. niger glukoamilasepromoter (glaA) geplaas. Die onderskeie virale gene is ook aan die koderende gedeelte vir die katalitiese domein van A. niger glukoamilase gelas om fusieproteïene te vorm. Glukoamilase word teen hoë vlakke deur Aspergillus vervaardig en uitgeskei en kan dus moontlik dien as draerproteïen om sekresie van die proteïne te bevorder. Transformasie van die geenkonstrukte na laboratoriumrasse van A. niger en A. awamori het verskeie transformante gelewer. Een A. niger transformant bevattende die S-geen onder transkripsionele beheer van die gpdA promoter het minstens sewe kopieë van die uitdrukkingskaset in sy genoom geïntegreer en het hepatitis B pseudovirale partikels intrasellulêr teen vlakke van 0.4 mg/I swamkultuur vervaardig. Hierdie vlakke is omtrent tienvoudig hoër as die vlakke van 0.01 - 0.025 mg/I wat S.cerevisiae oorspronklik opgelewer het. Nie een van die ander transformante het rekombinante S of L proteïene vervaardig nie en sekresie van virale proteïen kon nie getoon word nie. Hierdie verskynsel mag te wyte wees aan verskeie faktore insluitende vektor-kopiegetal, setel van integrasie en proteolitiese aktiwiteit. Die belangrikste insig uit hierdie studie aangaande sekresie van heteroloë virale proteïene is dat die koppeling van die virale omhulsel-proteïen aan 'n draerproteïen sekresie benadeel het, eerder as om dit te bevorder. Hierdie verskynsel is te wyte aan die inherente geneigdheid van virale omhulselproteïene om 'n kompleks te vorm. Die studie dien ook as "bewys van beginsel" dat Aspergillus wel 'n werkbare alternatiewe gasheer vir die produksie van hepatitis B pseudovirale partikels is, en dat dit verder ondersoek sou kon word as potensiële gasheer vir die heteroloë uitdrukking van ander virale proteïene.

published_or_final_version / Surgery / Master / Master of Philosophy

Viral hepatitis has been a universal human affliction for thousands of years. Only recently has it become understood, and there are still many unanswered questions. This dissertation examines the epidemiology of viral hepatitis in a county in the southwestern United States. An historical review traces the history of concepts of jaundice and details recent advances in the understanding of the transmission of the clinical entities grouped under the heading of viral hepatitis. Age specific incidence rates for all forms of hepatitis in the study population are compared to national rates. Data indicate that the study population experiences higher rates of enteric disease and lower rates of sexually transmitted disease than the nation as a whole. The hypothesis that diseases with similar routes of transmission will be associated with each other and show similar socioeconomic patterns was tested using three year average census tract incidence rates for 1982-84. In almost all samples, hepatitis A and shigellosis are more similar to each other than either is to hepatitis B. A similarity between hepatitis B and syphilis is also evident, but in fewer samples, reflecting their more disparate routes of transmission. Different relationships between incidence rates and socioeconomic variables are evident when the analysis is done using data from the fifty states for 1982. Participant observation as a disease investigator generated information on beliefs about hepatitis among lay and medical personnel. Often these beliefs diverge significantly from accepted facts about hepatitis. These findings have implications for the design of public health programs to control communicable diseases with similar modes of transmission.