The theophylline clearance was evaluated in patients with liver dysfunction and/or congestive heart failure. One hundred and twenty two patients were categorized into four groups; Group I: Liver dysfunction (n=20), Group II: Congestive heart failure (CHF, n=22), Group III: Both liver dysfunction and CHF (n=12), and Group IV: Control group (n=68). The severity of liver dysfunction and CHF were evaluated using Child-Turcotte- Pugh index (CTP) and a Cardiac Function index, respectively. Theophylline clearance was significantly decreased in Groups I, II, and III when compared to the control group; but, no significant difference was found among these three groups (mean values were 0.515, 0.479, 0.417, and 0.682 mllmin/kg, respectively). Moreover, patients with compensated cirrhosis or moderate to severe CHF had the lowest theophylline clearance values (mean values 0.344, and 0.335 ml/min/kg, respectively). There was a significant correlation between Cardiac Function index and theophylline clearance (r=-0.621) in Group II. Smokers had larger theophylline clearance values than those of nonsmokers in Groups I, II, and IV. Impairment of theophylline clearance did not correlate well with any of the indices of liver function or the CTP index. A model for prediction of the clearance in CHF was developed, which consisted of a Cardiac Function index and smoking habit. This model accounts for approximately 60% of the variation of theophylline clearance. However, models describing theophylline clearance in liver dysfunction and in congestive heart failure with liver dysfun ction did not appear to be useful. Thus, routine laboratory data and indices of liver function were not helpful in evaluating the impaired hepatic theophylline elimination. The Cardiac Function index appeared to be useful in estimation of theophylline clearance in CHF; however, the association between the theophylline clearance and severe CHF needs to be evaluated further.

No description available.

Hepatitis E virus (HEV) is an important human pathogen, with pigs and likely other animal species serving as natural reservoirs. There are currently four recognized HEV genotypes that infect humans within the genus Hepevirus of the family Hepeviridae. Genotypes 1 and 2 are human viruses that are associated with waterborne and fecal-oral transmission in developing countries, while genotypes 3 and 4 have been identified in humans and other animal species and are zoonotic and endemic in both industrialized and developing countries. In my dissertation research, we identified the first strain of HEV from rabbits in the United States. We subsequently determined the complete genome sequence of the virus. Phylogenetic analyses of the full-length sequence indicated that U.S. rabbit HEV is a distant member of the zoonotic genotype 3, thus raising a potential concern for zoonotic infection. In order to investigate the cross-species potential of rabbit HEV, we then determined its antigenic cross-reactivity with other animal strains of HEV. Additionally, we demonstrated that the novel rabbit HEV could cross species barriers and infect pigs under experimental conditions. Finally, we attempted to determine the risk factors and sources of foodborne HEV infection in the United States. We detected HEV for the first time from non-liver pork commercial products in the United States and demonstrated consumption of undercooked meat a risk factor for HEV infection. HEV sequences of genotype 3 origin were detected from pork products purchased from grocery stores in Southwest Virginia. Approximately 6.3% (21/335) of university students tested seropositive for HEV antibodies and, importantly, those with a history of consuming undercooked meats were 13 times more likely to be seropositive. These results further underscore the importance of cooking pork thoroughly and using proper hygiene when preparing meals. / Ph. D.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an emerging virus of global distribution. At least four distinct genotypes of HEV exist worldwide: genotype 1 and 2 HEV strains are responsible for waterborne epidemics; genotype 3 and 4 HEV strains are responsible for sporadic occurrences of acute hepatitis E. Genotype 3 and 4 HEVs are zoonotic and have a more expanded host range than genotypes 1 and 2 which are restricted to humans. Genotype 3 and 4 HEV isolates obtained from animal tissues are genetically very similar, or identical in some cases, to human HEV recovered from hepatitis E patients. The objectives of this dissertation research were to assess the zoonotic foodborne transmission of HEV and elucidate the viral determinants of HEV host range. To determine the risk of HEV foodborne transmission, 127 packages of commercial pig liver were tested for HEV RNA. Eleven percent of them were positive for HEV RNA and the contaminating virus remained infectious. We also demonstrated that medium-to-rare cooking condition (56°C) does not completely inactivate HEV, although frying and boiling of the contaminated livers inactivated the virus. To reduce the risk of foodborne HEV transmission, commercial pig livers must be thoroughly cooked for consumption. To determine the host range of genotype 4 HEVs, pigs were inoculated with a genotype 4 human HEV. All pigs developed an active HEV infection indicating that genotype 4 human HEVs can cross species barriers and infect pigs. To identify viral determinant(s) of species tropism, ORF2 alone or in combination with its adjacent 5′ junction region (JR) and 3′ non-coding region (NCR), were swapped between genotypes 1 and 4, 3 and 4, and 1 and 3 to produce 5 chimeric viruses. Chimeric viruses containing ORF2 or JR+ORF2+3' NCR from genotype 4 human HEV in the backbone of genotype 3 swine HEV were viable in vitro and infectious in vivo. Chimeric viruses containing the JR+ORF2+3'NCR of genotypes 3 or 4 HEV in the backbone of genotype 1 human HEV were viable in vitro but non-infectious in pigs, suggesting that ORF1 may also be important for host range. / Ph. D.

HEV is an important public health concern due largely to water-borne outbreak. Recent research confirms individual cases of zoonotic transmission due to human exposure to contaminated animal meats. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. In addition to humans, strains of HEV have been genetically identified from pigs, chickens, rats, mongoose, deer, rabbits and fish. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In the first study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. Sera from all pigs were tested for HEV RNA and IgG anti-HEV, and fecal samples were also tested for HEV RNA each week. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia and fecal virus shedding of challenge viruses were not detected, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenge with homologous and heterologous genotypes 3 and 4 HEV, respectively. Immunogenic epitopes are located within the open reading frame 2 (ORF 2) capsid protein and recombinant ORF 2 antigens are capable of preventing HEV infection in non-human primates and chickens. In the second study we expressed and purified N-truncated ORF 2 antigens based on swine, rat, and avian HEV strains. Thirty pigs were randomly divided into groups of 6 pigs each and initially vaccinated with 200µg swine ORF 2 antigen, rat ORF 2 antigen, avian ORF 2 antigen, or PBS buffer (positive and negative control groups) and booster with the same vaccine 2 weeks later. At 4 wks, after confirming seroconversion to IgG anti-HEV antibody with ELISA, all groups except the negative control were challenged with swine genotype 3 HEV (administered intravenously). The protective and cross-protective abilities of these antigens were determined following swine genotype 3 challenge by evaluating both serum and fecal samples for HEV RNA using nested RT-PCR and IgG anti-HEV using ELISA. The results from these two studies have important implications for future development of an effective HEV vaccine. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that selected goat sera were capable of neutralizing HEV in cell culture. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in 7 out of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the fecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains. / Ph. D.

Mathematical modeling of biological systems has improved the knowledge of scientists for many years. In virology, particularly in the study of hepatitis B virus, mathematical models were used to explain interactions between hepatitis B virus and the human host in the absence and presence of interventions such as drug therapy and vaccines. This thesis seeks to explain the role of e-Antigen, a particle produced by hepatitis B virus, in the pathogenesis of hepatitis B infection. To accomplish this goal, I will provide biological background as well as previous modeling work on the role of e-Antigen in hepatitis B virus infection, before finally developing a new model adapted specifically for connecting hepatitis B progression with e-Antigen and drug therapy. I will analyze the model both analytically and numerically, fit it to virus data from humans chronically infected with hepatitis B that undergo drug therapy, and draw conclusions about the relation between drugs, immune activation, and loss of e-Antigen. / Master of Science

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2005. / The development of non-replicating vaccines is an emerging option for safe, effective vaccines, several of which contain virus-like particles (VLPs). Many recombinant expression systems have been evaluated as hosts for VLP production for the prevention of infectious diseases. The filamentous fungi Aspergillus niger has emerged as a potential alternative expression system for cost effective VLP vaccine production. Hepatitis B surface antigen (HBsAg) was used as a model VLP product to benchmark A. niger’s production capacity with those of Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha. Bioprocessing strategies were used to optimise VLP production by recombinant A. niger in batch culture. In particular, the effect of the parameters culture temperature, inoculum concentration, agitation intensity, dissolved oxygen (dO2) concentration and culture pH on biomass formation, morphology and VLP (HBsAg) production concentration was quantified. At an optimum agitation of 100 rpm and optimum dO2 concentration of 50 %, HBsAg production levels were increased 9-fold compared to yields obtained in shakeflask cultivation. Highest HBsAg production levels of 3.6 mg.ℓculture -1 and 350 μg.gDW -1 were recorded, at a biomass concentration of 10.5 gDW.ℓculture -1. These production levels compare favourable with those obtained by other production systems under similar conditions. HBsAg VLPs mostly accumulated intracellularly, although under optimum bioreactor conditions significant HBsAg accumulation in the cytoplasm and culture supernatant was also observed. The impact of these process parameters on VLP production and cell morphology was attributed to environmental stress conditions. Volumetric biomass and HBsAg production levels were maximised under conditions of lowest environmental stress, resulting in the most optimal small-pelleted morphology. These results indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.

published_or_final_version / Dentistry / Master / Master of Dental Surgery

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

Se estudiaron 236 historias clínicas de pacientes atendidos en el Hospital Edgardo Rebagliati Martins, desde el año 2000 hasta julio del 2004.con fibrosis o cirrosis hepática por hepatitis viral B y C con el objetivo de evaluar el valor de diagnóstico de la ultrasonografía en comparación con el diagnóstico histopatológico. Se aplicó un sistema de puntaje ultrasonográfico a cada paciente de acuerdo a los signos US clasificándolos en una escala de 4-11. Se transcribieron los puntajes histopatológicos según el sistema METAVIR y se evaluó la sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo para la puntuación US, compuesta de superficie, parénquima, borde hepático y pared porta. A partir del punto óptimo se hallaron gráficos de curvas ROC lo que permitió tener una mejor visión de las pruebas de diagnostico. Se halló que una puntuación US 6 fue el mejor punto de corte para la predicción de Cirrosis y fibrosis grado III relacionados a HBV y HCV, con sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de 97.2%, 71.4%, 89.9%, 90.95 respectivamente. Las puntuaciones US están altamente y significativamente correlacionado 0.73(P<0.01) con la histopatología. La ultrasonografía en el diagnostico de la fibrosis y cirrosis por hepatitis B y C, muestra un mayor valor en la sensibilidad (85.39), la especificidad (85.71) y exactitud (85.59) en relación a la superficie del hígado. Los resultados demuestran que la ultrasonografía es un examen confiable para el diagnostico de la cirrosis y la fibrosis hepática tanto para la hepatitis viral B y C, aunque con mayor predicción para la hepatitis viral B. Los datos permiten concluir que la fibrosis y la cirrosis por hepatitis B y C, pueden ser correctamente diagnosticadas con un 85.59% de exactitud empleando un solo signo ultrasonográfico (superficie del hígado).