Dissertations / Theses on the topic 'Liver disease; Immunology'

Liver cirrhosis is the fifth leading cause of death worldwide and the definitive treatment for liver cirrhosis is liver transplantation although there are limitations such as organ availability and surgical risks. Therefore, alternative therapies have been studied extensively and stem cell therapies have shown some promising results although most studies are small and not randomised. The aim of this thesis was to explore the effectiveness of stem cell therapy in patients with chronic liver disease as well as explore the mechanism behind fibrosis resolution achieved with cell therapy. There were three parts to the thesis: firstly, I examined the mechanistic actions behind fibrosis reduction by the infusion of bone marrow derived haematopoietic stem cells (HSC) in mice chronic fibrosis liver injury model. I worked on both immune-histochemical staining and qPCR to measure the effect oval cell response, matrix metalloproteinases and macrophage subsets within the liver with HSC therapies. Secondly, I recruited patients with chronic liver diseases for a multicentre, randomised, controlled trial to assess the clinical effectiveness of either subcutaneous granulocyte-colony stimulating factor (GCSF) or GCSF with repeated HSC infusions. The co-primary outcomes were improvement in severity of liver disease measured by model for end stage live disease (MELD) at 3 months and the trend of MELD change over time. The results showed that neither of the treatments improved the clinical outcomes. Lastly, I performed a systematic review of current published studies of stem cells therapies in liver diseases. The results showed that stem cells improved patients’ clinical parameters in the short term (< 6 months) but had no benefit on long term outcomes. In conclusion, bone marrow derived stem cell therapy did not seem to be effective in liver cirrhosis.

CD161 γδ T-cells have been implicated in the pathogenesis of Multiple Sclerosis however their role in health and chronic liver disease requires further exploration. In health, the majority of γδ T-cells expressed CD161 – a C-type lectin, and predominantly expressed the Vδ2 chain. The CD161+ γδ T-cells demonstrated a Th1-like pattern, expressing IFN-γ, TNF-α and Granzymes/Perforin when compared to the CD161- subset. The CD161+ γδ T-cells also expressed CCR6 and IL-18R thus also displaying a Th17-like pattern. These cells were also found in the lamina propria in the gut and rapidly expanded in the 1^st few weeks of life in the periphery. On gene array analysis, there were 409 genes expressed on the CD161+ γδ T-cells when compared to their CD161-ve counterparts including those coding for β2 receptors, CCL20, Acetycholinesterase, CCR1 and IL-18R. A potential clinical correlation to cardiac diseases was found when the upregulated genes were analysed. When the CD161+ γδ and CD161+ αβ T-cell populations were compared via gene-array, an association with a risk variant for coeliac disease was found. Thus in health, CD161+ γδ T-cells are not only a distinct subset of T-cells (confirmed by a FACS approach and gene array methods), but also the expression of CD161 may be linked to common genetic signals downstream in cell processes and disease pathogenesis, irrespective of T-cell subset population. In chronic liver disease there was a significant reduction in the periphery of CD161+ γδ T-cells in patients with chronic Hepatitis C (HCV) and an increase in patients with Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis when compared with healthy individuals. The CD161+ γδ T-cells appeared to be of a different phenotype in HCV infection. There was no overall significant localisation into of CD161+ γδ T-cells patients with chronic liver disease or specifically in HCV infection. There was however a CD161+ γδ T-cell enrichment in the liver in patients with Non-Alcoholic Fatty Liver disease. The CD161+ γδ T-cells were also found in Hepatocellular Carcinoma tissue. Overall it appears the CD161+ γδ T-cells are indeed a unique subset, playing a distinct role in health, as part of an early innate response, but also potentially involved in disease pathogenesis.

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression. My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

Nonalcoholic fatty liver disease (NAFLD) has become one of the most common hepatic diseases worldwide, making the diagnosis and management of NAFLD an emerging public health issue. Theories associated with NAFLD surmise that inflammation may be the root cause, along with the complex interplay of other chronic conditions such as obesity, metabolic syndrome, diabetes, dyslipidemia, and cardiovascular disease (CVD). It is unknown if other inflammatory conditions such as rheumatoid arthritis (RA), along with the use of methotrexate (MTX), might confer increased risk for NAFLD. Longitudinal data collected from a retrospective cohort of 17,481 adult RA patients in the United States were used to determine the incidence and factors associated with the development of NAFLD using a noninvasive tool (Fibrosis-4 score). Results of the Kaplan Meier analysis showed that 31% of this cohort developed NAFLD, in about 7 years from baseline, with most having mild to moderate disease and only 1.4% with advanced disease. RA patients also had a prevalence of chronic conditions associated with NAFLD, as seen in the general population. In the Cox proportional hazard multivariate analysis, age (middle and elderly), hypertension, CVD, dyslipidemia, metabolic syndrome, exercise, use of MTX, and non-MTX antirheumatic drugs were independent predictors for the development of NAFLD. This research could improve early diagnosis of NAFLD using a novel noninvasive tool. Increase awareness of the prevalence and causes of NALFD inform clinical practice and management of the disease and influence policy about this chronic condition in patients with RA.

Liver disease is the fifth largest killer in the United Kingdom and with the numbers of diagnoses increasing each year there is an urgent need for novel therapeutic interventions. This thesis examines one potential target, Vascular Adhesion Protein (VAP)-1: an amine oxidase enzyme with reported adhesin functionality. The results herein confirm a primarily sinusoidal localisation of VAP-1, expression of which was upregulated in the liver during chronic inflammation correlated with diminished enzyme activity. Functional analysis of sinusoidal VAP-1 in vitro did not demonstrate any effect of inhibition on leukocyte recruitment, unlike that observed in other tissues. Furthermore, only neutrophils were capable of binding to recombinant VAP-1 under flow conditions. Further investigation highlighted the importance of this intimate relationship during neutrophil-endothelial interactions; revealing the first evidence that neutrophils also express catalytically active VAP-1. Neutrophil effector functions, such as the formation of extracellular traps, were also hindered by recombinant VAP-1. This was also observed in wild-type mice but not those expressing a catalytically inactive form of VAP-1 (SSAOKO). Following acute injury, these mice also exhibited expanded intrahepatic macrophage and NKT cell populations compared to control. In combination, these data highlight the complex role that VAP-1 plays during inflammatory liver disease.

This thesis addresses the genetic basis of a rare autosomal recessive primary immunodeficiency disorder with the characteristic additional feature of venoocclusive disease of the liver (VODI). The interest in this condition was stimulated both by the potential to identify the genetic basis of a rare immunodeficiency and the opportunity to gain an insight into the biological basis of hepatic veno-occlusive disease, a poorly understood condition that is encountered most frequently in Australia as a consequence of bone marrow transplantation. The gene responsible for VODI was identified by homozygosity mapping and DNA sequence analysis of positional candidates and was shown to be the PML Nuclear Body expressed protein Sp110. This is the first time a PML Nuclear Body protein has been shown to be involved in immunodeficiency disorder. Subsequent immunofluorescence studies of affected patient cell lines showed absence of Sp110 in patient B cells. The role of SP110 alleles in the susceptibility of bone marrow transplant patients to hepatic veno-occlusive disease was investigated using a cohort of patients from the Fred Hutchinson Cancer Center, Seattle. A SNP association study identified initial evidence for an association, but the study lacked sufficient power after correction for multiple testing. Contemporaneously, Dr Igor Kramnik published a report that the murine homologue of Sp110, Ifi75 (also termed Ipr1) was deleted in mice that were supersusceptible to infection with Mycobacterium tuberculosis. A further SNP association study was therefore performed utilising a NSW cohort of Mantouxpositive South East Asian migrants, which detected evidence that alleles of SP110 may be associated with progression of M. tuberculosis infection. Again, the limited size of this cohort precluded definitive findings.

First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway. First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway.

The development of an efficacious P. falciparum malaria vaccine remains a top priority. Pre-erythrocytic vaccine efforts have traditionally focussed on two well- known antigens, CSP and TRAP, yet thousands of antigens are expressed throughout the liver-stage. The work described in this thesis aimed to assess the ability of other pre-erythrocytic antigens to induce an immune response and provide protective efficacy against transgenic parasites in a mouse model. Research undertaken in our laboratory has demonstrated the ability of prime-boost viral vectored sub-unit vaccination regimens to elicit high levels of antigen-specific T cells. Eight candidate antigens were therefore expressed individually in the viral vectors ChAd63 and MVA. Two antigens, PfLSA1 and PfLSAP2, were identified that confer greater protective efficacy in inbred mice than either CSP or TRAP. PfLSA1 was also able to induce almost complete sterile efficacy in outbred mice, suggesting this vaccine should be assessed in a clinical trial. Immune responses to the candidate antigens were also assessed in human volunteers following their first exposure to controlled malaria infection. The antigen TRAP was further characterised by epitope mapping in volunteers vaccinated with ChAd63-MVA ME-TRAP. However, no functional T cell assay exists to measure inhibition of P. falciparum liver-stage parasites. An improved murine in vitro T cell killing assay was developed, and preliminary experiments were conducted that demonstrate the potential and promise of a P. falciparum T cell killing assay. Such assays will not only allow mechanistic studies to be undertaken, but could also change the way we screen pre-clinical liver-stage vaccines.

Chronic hepatitis C virus (HCV) infection exhibits persistent high viral load, inducing T cells differentiation and dysfunction in chronically infected individuals. Recent longitude studies in both HCV specific- and bulk T cells reveal that chronic immune stimulation is the driving force for the impaired T cell functions, however, the underlying mechanisms remain elusive. Here, we show that peripheral CD4+ T cells from chronically HCV-infected patients exhibit lymphopenia with the reduction of naïve population and expansion of effector memory T cells. CD4+ T cells from HCV patient also display elevated activation markers. including HLA-DR, GLUT1, Granzyme B, and short-lived effector marker CD127- KLRG1+, whereas stem cell-liked transcription factor TCF1 and telomere sheterin subunit TRF2 are significant reduced, comparing to age- and gender-matched healthy controls. Mechanistically, ex vivo T cell differentiation revealed that CD4+ T cells from HCV patients exhibit PI3K/Akt/mTOR signaling hyperactivation upon TCR stimulation, favoring pro-inflammatory effector differentiation with TRF2 downregulation, rendering telomere dysfunction induced foci (TIFs) accumulation, resulting in telomeric DNA damage and cellular apoptosis. Importantly, exacerbation of telomere deprotection by knockdown of TRF2 expression in healthy T cells resulted in an increase in telomeric DNA damage and T cell apoptosis; whereas overexpression of TRF2 in HCV-T cells led to an alleviation of telomeric DNA damage and T cell death. Additionally, inhibition of Akt signaling during T cell activation can preserve precursor memory population, while limiting inflammatory effector expansion, DNA damage, and cell death. Taken together, these results suggest that modulation of immune activation by inhibiting Akt signaling and protection of telomeres by enforcing TRF2 expression could open new therapeutic strategies to balance adaptive immune responses in the setting of chronic immune activation and inflammatory in vulnerable populations such as chronically viral infected individuals.

Enigmatic amphibian declines are strongly associated with infectious diseases, including chytridiomycosis, caused by the fungus 'Batrachochytrium dendrobatidis' (Bd), and ranaviral disease induced by ranaviruses. A series of data analyses, field sampling, laboratory experimentation and molecular techniques were utilized to test the hypothesis that the international live animal trade is contributing to the spread of these amphibian pathogens. Regions sampled included specific locations in North America, South America and Asia. The magnitude of the live importation of amphibians into the United States (U.S.) was calculated in the millions of animals per year. Batrachochytrium dendrobatidis and ranavirus infections were identified in live food animals in: (a) U.S. wet markets (in 'Lithobates catesbeianus', the North American bullfrog); (b) frog farms in Taiwan (in 'L. catesbeianus'); and (c) wet markets in Taiwan (in 'Rana tigrina', the Chinese bullfrog). 'Batrachochytrium dendrobatidis' infections were also identified in 'L. catesbeianus' farmed in Brazil and in 8 species of amphibians in the U.S. pet trade (in captive bred and directly imported animals). Detection of Bd in individuals upon initial entry into the U.S. demonstrated definitively the trans-continental movement of this potentially lethal amphibian pathogen. Laboratory experimentation illustrated transmission of Bd from a highly traded carrier host ('L. catesbeianus') to frogs of the same species, and to a known susceptible frog species ('Litoria caerulea', White's tree-frog). Molecular data linked U.S. market frogs and their associated ranaviral infections to origins in Asia and South America. Genotyping of pure, cultured isolates of Bd from farmed 'L. catesbeianus' in Brazil showed that they were most similar to isolates from native amphibians in Latin America when compared to a global dataset, providing evidence for transmission between wild and farmed amphibians. Analysis of isolates from market 'L. catesbeianus' led to the discovery of a novel genotype of Bd in Ypsilanti, Michigan that appeared to have a disparate lineage compared with the previously identified panzootic genotypes. DNA sequence analysis from the Ypsilanti frog indicated its origin in Brazil, where the novel genotype was also discovered in native Brazilian frog populations. The demonstration of amphibian pathogens on frog farms, in live wet markets and at border crossings, the evidence of inter- and intra-species transmission, and the existence of a novel genotype in traded anurans, all indicate that international transport of amphibians and their pathogens could produce not only mixing of pathogen genotypes, but the inadvertent spread of strains of unknown virulence that could negatively impact amphibian health. In 2008, data from research contained in this thesis were instrumental in the decision to list both Bd and ranaviral disease in the World Organization for Animal Health Aquatic Animal Health Code, providing guidelines (such as quarantine procedures) to limit the spread of these pathogens through the trade. The evidence provided in this work re-enforces the need for urgent action to minimize the potential spread of amphibian diseases through international trade routes, for the benefit of amphibian populations throughout the world.

Les maladies du foies non-alcoholiques (NAFLD) représentent un large spectre de pathologies qui comprennent la stéatose ou la stéatohepatite non alcoolique (NASH). Avec un nombre de patients diagnostiqué en constante augmentation, les NAFLD sont devenues un problème majeur de santé publique dans le monde. Un hypothése de « multiples hits » à été décrite pour regrouper l’ensemble des dérégulations métaboliques associées à la transition de la stéatose vers la NASH. Cette étape est critique au cours de la pathogénèse des NAFLD. En effet dans le cas où la NASH n’est pas prise en charge, elle peut se transformer en une fibrose ou une cirrhose et finalement en un cancer hepatocellulaire (CHC). Ainsi, l’analyse des évènements précoces au cours des NAFLD sont essentiels pour comprendre son évolution vers des pathologies plus dommageables. Dans le foie, plusieurs populations de cellules sont impliquées dans le métabolisme et les fonctions hépatiques ainsi que dans la surveillance immunitaire. Parmi celles ci, le groupe 1 ILC est enrichie dans le foie et peut rapidement induire une réponse immunitaire en produisant des cytokines ou en induisant la mort cellulaire. Le groupe 1 ILC hépatique est composé des cellules NK (Natural Killers) et des ILC1 (Cellules Lymphoïdes Innées de type 1), deux populations de cellules qui partagent un phénotype semblable. Cependant, elles forment bien deux lignages distincts avec des caractéristiques uniques. Nous proposons donc d ‘étudier le(s) role(s) des cellules NK et des ILC1 au cours des étapes précoces des NAFLD.Dans cette étude, nous avons démontré les cellules NK et les ILC1 subissent des modifications hétérogènes au cours des étapes précoces des NAFLD. Alors que les ILC1 présentent une diminution de leurs marqueurs d’activation et d’inhibition et de production de granzyme B, nous détectons une accumulation de cellules NK produisant de l’interféron gamma (IFNg). Ces modifications sont induites rapidement au cours de la stéatose et sont même antérieures au recrutement et à l’activation d’autres cellules immunitaires du foie. Nous soulignons également l’importance du système immunitaire intestinal au cours de l’inflammation hépatique et proposons que la lamina propria de l’intestin puisse servir de réservoir de cellules NK au cours de ce processus. Finalement, nous avons démontré que l’IFNg, secrétée entre autre par les cellules NK induise des dommages au cours de la stéatose mais n’est pas impliquée dans le recrutement ou les modifications observées sur le groupe 1 ILC. Cette étude apporte de nouveaux éléments sur les étapes précoces des NAFLD ainsi que le rôle du groupe 1 ILC au cours de l’inflammation hépatique. Cette étude souligne également l’importance de la distinction entre les cellules NK et les ILC1 au cours des pathologies du foie
Non-alcoholic fatty liver diseases (NAFLD) is a spectrum of liver pathologies that encompass diseases such as steatosis or non-alcoholic steatohepatitis (NASH). With a constant increase of patients diagnosed, NAFLD is becoming a major concern of public health worldwide. A “multiple hits” hypothesis has been described to regroup the metabolic disorders that are associated with the transition from steatosis to NASH. This transition is a critical step during NAFLD pathogenesis as untreated NASH can further develop into fibrosis, cirrhosis and ultimately to hepatocellular carcinoma (HCC). Thus, the analysis of early events occurring during during NAFLD is critical to understand its evolution to more severe pathologies. In the liver, diverse cell populations are involved in hepatic metabolism, function and immune surveillance. Among them, the group 1 ILC is enriched in the liver and can quickly induce an immune response by producing cytokines or inducing cell death. Hepatic group 1 ILC is composed of Natural Killer (NK) cells and Innate Lymphoid Cells 1 (ILC1), two cell populations that share a similar phenotype. Nevertheless they constitute two distinct cell lineages that have unique features. Here we propose to study the roles of NK cells and ILC1 during the early stages of NAFLD.In this work, we demonstrated that NK cells and ILC1 diverge in phenotype and function during the early stages of NAFLD pathogenesis. While ILC1 showed a down-regulation of inhibitory markers and down-regulation of granzyme B, we detected an increase of interferon gamma (IFNg) secreting NK cells. These modifications were found shortly after the induction of steatosis and preceded other hepatic immune cell recruitment or activation. Our work highlighted the role of the immune intestinal populations during liver inflammation and identified the intestinal lamina propria as a potential source of NK cells during this process. Finally, we demonstrated that IFNg is inducing liver damage, but is not involved in hepatic group 1 ILC recruitment or modification in our model of steatosis.This study brings new insights on the early events of NAFLD and the role of hepatic group 1 ILC during liver inflammation. It also underlines the importance of distinguishing the roles of NK cells and ILC1 in liver pathologies

Master of Veterinary Biomedical Sciences
Department of Clinical Sciences
Bradley White
Objective- To compare serologic response and viral replication following intranasal administration of a modified-live bovine rhinotracheitis (IBR) parainfluenza-3 (PI-3) vaccine in high (32°C) and moderate (21°C) ambient temperatures. Animals- 28 heifers (mean body weight, 206.8 kg) Procedures- Heifers randomly allocated to treatment groups: High Ambient Temperature (HAT, n=10): received vaccine, housed outdoors, Moderate Ambient Temperature (MAT, n=10): received vaccine, housed indoors, High Ambient Control (HAC, n=4): no vaccine, housed outdoors, Moderate Ambient Control (MAC, n=4): no vaccine, housed indoors. Rectal and nasal mucosal temperatures were recorded every 2 hours from 8am to 8pm on trial days 0 and 1. Nasal swabs were collected on trial days 0 through 7 for virus isolation. Serum samples were collected for serology on trial days 0, 7, 14, and 28. Results- Rectal temperatures did not differ among treatment groups over the study period, but nasal temperatures were higher in the HAT calves compared to MAT group at study hours: 6, 24, 30, 32, and 38. Two weeks post-vaccination, IBR titers were significantly greater in vaccinates (HAT,MAT) relative to non-vaccinates (HAC, LAC), but no differences were identified among HAT and MAT. Viable IBR virus was recovered via virus isolation from all vaccinated calves (HAT,MAT) on trial days 1 through 6. Conclusions and Clinical Relevance- The ability to isolate IBR and stimulate the calf immune response following administration of a modified-live IBR-PI3 intranasal vaccine did not differ in calves housed in temperature-controlled and high ambient temperature environments.

A study of the causes of liver enlargement amongst black patients at King Edward VIII Hospital, Durban, South Africa has revealed that congestive cardiac failure (36.7%), amoebic liver abscess (7.1%), hepatocellular carcinoma (5.8%) and cirrhosis (5.4%) are the most common causes in this population. Liver biopsy was needed to determine the cause in 28.7% of patients studied. The diagnostic yield of percutaneous liver biopsy was increased by obtaining 2 or 3 consecutive specimens for histological examination by redirecting the biopsy needle through a single entry site. This benefit was achieved without an increase in morbidity or mortality. Fatalities and complications associated with liver biopsy were more frequent at this hospital than in hospitals in Europe, The United Kingdom and North America. The complication rates after percutaneous or peritoneoscopic biopsy were 2.0% and 2.3% respectively. A total of 6 deaths was recorded. The morbidity and mortality rates were not increased when more than one specimen was taken during percutaneous biopsy. In the majority of patients in whom biopsy was carried out, after-care was either non-existent or inadequate. The "Tru-Cut" needle was used for all percutaneous liver biopsies at King Edward VIII Hospital. Two techniques, including the method recommended by the manufacturer, have been found to be incorrect; the needle must be used correctly if an adequate biopsy specimen is to be obtained for histological examination and if serious complications are to be avoided. Hepatic tuberculosis was diagnosed in 9% of patients with unexplained hepatomegaly who were subjected to liver biopsy. This disease did not yield any consistent clinical findings. In addition, liver function tests were of little diagnostic value and results of hepatic imaging techniques were often normal. Accordingly, a high index of suspicion is needed and liver biopsy is essential in patients with unexplained hepatomegaly or hepatospienomegaly, or pyrexia of unknown origin since biopsy provides the only means of diagnosing hepatic tuberculosis. The accuracy of both ultrasonography and scintigraphy in distinguishing between normal and diseased livers was low (68% and 74% respectively). These techniques performed better at detecting focal than diffuse liver disease; the sensitivity of ultrasonography and scintigraphy in focal and diffuse disease were 88% and 92%, and 27% and 54% respectively. The specificity of both procedures was high for both types of liver disease (range 91-96%). Overlap between the ultrasonographic features of amoebic liver abscess, hepatocellular carcinoma and metastatic carcinoma resulted in a correct final diagnosis being made in only 81% of patients with amoebic liver abscess, 29% with hepatocellular carcinoma and 43% of patients with metastatic carcinoma who had an ultrasound scan. Neither technique was capable of determining the cause of diffuse liver disease. Therefore, when diffuse parenchymal liver disease is suspected, liver biopsy is needed to determine the presence and nature of the disease. In addition, liver biopsy or aspiration is usually required to determine the cause of focal disease in selected patients in whom space-occupying lesions are detected on hepatic imaging studies.
Thesis (M.D.)-University of Natal, Durban, 1990.

by Lau Tze Chin, Gene.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1991.
Includes bibliographical references (Leaves 170-195).
Abstract --- p.1
Acknowledgement --- p.3
List of tables --- p.4
List of figures --- p.6
List of abbreviations --- p.7
Chapter Chapter One - --- Introduction --- p.9
Chapter 1.1. --- Historical Aspects --- p.9
Chapter 1.2. --- Classification of hepatitis B virus --- p.12
Chapter 1.2.1. --- Hepadnaviruses --- p.12
Chapter 1.2.2. --- Comparative properties of hepadnaviruses --- p.13
Chapter 1.2.2.1. --- Physical properties --- p.13
Chapter 1.2.2.2. --- Genetic relatedness --- p.15
Chapter 1.2.2.3. --- Pathogenesis --- p.16
Chapter 1.3. --- Structural and morphological properties of HBV --- p.17
Chapter 1.4. --- Molecular biology of HBV --- p.20
Chapter 1.4.1. --- Molecular structure of HBV --- p.20
Chapter 1.4.1.1. --- Biochemistry of the virion envelope --- p.20
Chapter 1.4.1.2. --- The nucleocapsid --- p.21
Chapter 1.4.1.3. --- Structural features of HBV genome --- p.23
Chapter 1.4.2. --- Genetic organization of HBV --- p.24
Chapter 1.4.3. --- Infection cycle of HBV --- p.29
Chapter 1.4.3.1. --- Viral attachment and internalization --- p.29
Chapter 1.4.3.2. --- Replication of HBV --- p.30
Chapter 1.4.3.3. --- Gene expression and regulation --- p.31
Chapter 1.4.3.4. --- Host-virus DNA interaction --- p.33
Chapter 1.5. --- Epidemiology and transmission of HBV --- p.34
Chapter 1.5.1. --- World wide prevalence --- p.35
Chapter 1.5.1.1. --- HBsAg prevalence --- p.35
Chapter 1.5.1.2. --- Cumulative rate of HBV infection --- p.35
Chapter 1.5.1.3. --- Age specific pattern of HBV infection --- p.36
Chapter 1.5.2. --- Epidemiological pattern of HBV in Hong Kong --- p.37
Chapter 1.5.3. --- Mode of transmission --- p.38
Chapter 1.6. --- Clinical outcomes of HBV infection --- p.38
Chapter 1.6.1. --- Acute infection --- p.41
Chapter 1.6.2. --- Chronic infection --- p.42
Chapter 1.6.3. --- Primary hepatocellular carcinoma --- p.43
Chapter 1.7. --- Laboratory diagnosis of hepatitis B --- p.44
Chapter 1.7.1. --- The HBV markers --- p.47
Chapter 1.7.1.1. --- HBsAg and anti-HBs --- p.47
Chapter 1.7.1.2. --- HBcAg and Anti-HBc --- p.47
Chapter 1.7.1.3. --- HBeAg and anti-HBe --- p.49
Chapter 1.7.1.4. --- HBV-associated DM polymerase --- p.49
Chapter 1.7.1.5. --- HBV-DNA --- p.49
Chapter 1.7.2. --- Methodology in the detection of hepatitis B markers --- p.50
Chapter 1.7.2.1. --- Direct detection of HBV and HBV antigens --- p.50
Chapter 1.7.2.2. --- Serological detection of HBV markers --- p.51
Chapter 1.7.2.3. --- HBV-associated DNA polymerase assay --- p.51
Chapter 1.7.2.4. --- Molecular technique for the detection and quantitation of HBV-DNA --- p.52
Chapter 1.8. --- Antiviral therapy in hepatitis B --- p.52
Chapter 1.8.1. --- Therapeutic agents for treatment of HBV infection --- p.53
Chapter 1.8.1.1. --- Steroids --- p.53
Chapter 1.8.2.2. --- Nucleoside analogs --- p.54
Chapter 1.8.1.3. --- Interferon --- p.55
Chapter 1.8.2. --- Clinical trials of interferons --- p.55
Chapter 1.9. --- Extrahepatic tissue tropism of HBV --- p.62
Chapter 1.10. --- Objective and design of study --- p.65
Chapter 1.10.1. --- Objectives of study --- p.65
Chapter 1.10.2. --- Study design --- p.66
Chapter 1.10.2.1. --- Cross-sectional study --- p.67
Chapter 1.10.2.2. --- Longitudinal study --- p.67
Chapter 2.1. --- Materials --- p.71
Chapter 2.1.1. --- Patients recruitment and clinical materials --- p.71
Chapter 2.1.1.1. --- Cross-sectional study --- p.71
Chapter 2.1.1.2. --- Longitudinal study --- p.71
Chapter 2.1.2. --- Bacteria] stock --- p.71
Chapter 2.1.3. --- "Chemicals, equipments and consumables" --- p.72
Chapter 2.1.4. --- Buffers and solutions --- p.72
Chapter 2.1.4.1. --- Phosphate buffer saline (PBS) --- p.72
Chapter 2.1.4.2. --- Leucocyte lysis buffer (X 5)(LLB) --- p.72
Chapter 2.1.4.3. --- Buffer equilibrated phenol (BEP) --- p.76
Chapter 2.1.4.4. --- Phenol-Chloroform mixture --- p.76
Chapter 2.1.4.5. --- 3.0M sodium acetate (pH 5.2) --- p.76
Chapter 2.1.4.6. --- Tris-EDTA buffer (pH 8.0) (TE) --- p.76
Chapter 2.1.4.7. --- Stock salmom sperm DNA solution --- p.77
Chapter 2.1.4.8. --- Tracking dye --- p.77
Chapter 2.1.4.9. --- Tris-borate electrophoresis buffer (TBE) --- p.77
Chapter 2.1.4.10. --- Luria-Bertani Broth (LB) --- p.77
Chapter 2.1.4.11. --- Solution ] --- p.78
Chapter 2.1.4.12. --- Solution ]] --- p.78
Chapter 2.1.4.13. --- Potassium acetate buffer (pH 5.4) --- p.78
Chapter 2.1.4.14. --- Column elution buffer (CEB) --- p.78
Chapter 2.1.4.15. --- NPMEB solution --- p.79
Chapter 2.1.4.16. --- Neutralizing solution --- p.79
Chapter 2.1.4.17. --- Standard saline citrate (SSC) --- p.79
Chapter 2.1.4.18. --- Denhardt solution --- p.79
Chapter 2.1.4.19. --- Prehybridization solution (PS) --- p.80
Chapter 2.1.4.20. --- NETFAP Solution --- p.80
Chapter 2.1.4.21. --- Heparin solution --- p.81
Chapter 2.1.4.22. --- Hybridization mix for oligo-nucleotide probe --- p.81
Chapter 2.1.4.23. --- NEPS solution (pH 7.0) --- p.81
Chapter 2.1.4.24. --- Restriction endonuclease and buffer --- p.82
Chapter 2.2. --- Methods --- p.82
Chapter 2.2.1. --- Sample preparations --- p.82
Chapter 2.2.1.1. --- Isolation of plasma and peripheral blood leucocytes (PBL) --- p.82
Chapter 2.2.1.2. --- Extraction of DNA from Peripheral blood leucocytes --- p.83
Chapter 2.2.1.3. --- Quantitation of Peripheral blood leucocyte DNA --- p.83
Chapter 2.2.2. --- Preparation of radio-labelled HBV-DNA probe --- p.84
Chapter 2.2.2.1. --- Plating and selection of bacterial stock --- p.84
Chapter 2.2.2.2. --- Growth of E. coli HB101 and amplification of pAM6 --- p.84
Chapter 2.2.2.3. --- Harvesting of E. coli and extraction of plasmid pAM6 --- p.84
Chapter 2.2.2.4. --- Purification of plasmid pAM6 --- p.86
Chapter 2.2.2.5. --- Large scale isolation and purification of HBV genome from plasmid pAM6 --- p.86
Chapter 2.2.2.6. --- Radio-labelling of HBV-DNA --- p.88
Chapter 2.2.2.6.1. --- Nick-translation of total HBV-DNA genome --- p.88
Chapter 2.2.2.6.2. --- Multi-primer labelling of total HBV- DNA genome --- p.88
Chapter 2.2.2.6.3. --- End-labeling of 21-base HBV oligo- nucleotide --- p.88
Chapter 2.2.2.6.4. --- Determination of labelling efficiency --- p.89
Chapter 2.2.2.7. --- Purification of labelled HBV-DNA probe --- p.90
Chapter 2.2.2.7.1. --- Total genomic HBV-DNA probe (pAM6 probe) --- p.90
Chapter 2.2.2.7.2. --- Oligo-nucleotide HBV-DNA probe (oligo probe) --- p.90
Chapter 2.2.3. --- Hybridization study of clinical samples --- p.91
Chapter 2.2.3.1. --- Solution hybridization of sera samples --- p.91
Chapter 2.2.3.2. --- Spot hybridization of sera samples --- p.91
Chapter 2.2.3.2.1. --- "Pre-hybridization treatment of sera samples (adapted from Lin et al.,1987)" --- p.91
Chapter 2.2.3.2.2. --- Pre-hybridization and hybridization of the membrane --- p.92
Chapter 2.2.3.2.3. --- Washing of membrane --- p.92
Chapter 2.2.3.2.4. --- Final treatment and autoradiography: --- p.92
Chapter 2.2.3.3. --- Quantitation of HBV-DNA in the sera samples: --- p.93
Chapter 2.2.4. --- Assay for serological Hepatitis B marker --- p.93
Chapter Chapter Three - --- Results --- p.93
Chapter 3.1. --- Preparation of HBV-DNA probes --- p.95
Chapter 3.2. --- Radiolabelling of HBV-DNA --- p.95
Chapter 3.3. --- Hybridization methodology --- p.98
Chapter 3.4. --- Comparison of the performance of HBV-DNA probes --- p.100
Chapter 3.4.1. --- Quantitation of serum HBV-DNA --- p.100
Chapter 3.4.2. --- Comparative hybridization performance of different HBV-DNA probes --- p.105
Chapter 3.5. --- Clinical application of HBV-DNA probe:Detection of HBV-DNAin serum and peripheral blood leucocytes (PBL) --- p.109
Chapter 3.5.1. --- Cross-sectional study --- p.112
Chapter 3.5.1.1. --- Frequency of HBV-DNA detection in relation to different clinical manifestations --- p.112
Chapter 3.5.1.2. --- Frequency of HBV-DNA detection in relation to the serological status --- p.114
Chapter 3.5.1.3. --- Distribution of serum and PBL HBV-DNA level in chronic hepatitis B patients in relation to the different HBV-related manifestations --- p.119
Chapter 3.5.2. --- Longitudinal study of patients with chronic hepatitis B under interferon therapy with prednisolone pretreatment --- p.123
Chapter 3.5.2.1. --- Features of patients under study --- p.123
Chapter 3.5.2.2. --- Correlation between the occurrence of HBV- DNA and HBeAg in serum --- p.123
Chapter 3.5.2.3. --- Outcome of clinical trial: --- p.126
Chapter 3.5.2.3.1. --- Number of patients responding to therapy: --- p.126
Chapter 3.5.2.3.2. --- Variation in serum HBV markers during the course of study --- p.128
Chapter 3.5.2.3.3. --- Change of HBV-DNA statusin peripheral blood leucocytes --- p.134
Chapter Chapter Four - --- Dicussion --- p.140
Chapter 4.1. --- Preparation of HBV-DNA hybridization probes --- p.140
Chapter 4.1.1. --- Source of HBV-DNA --- p.140
Chapter 4.1.2. --- Raidolabelling of HBV-DNA --- p.141
Chapter 4.2. --- Hybridization methodology --- p.141
Chapter 4.2.1. --- Optimization of hybridization conditions --- p.141
Chapter 4.2.2. --- Comparison of the performance among different HBV- DNA probes --- p.144
Chapter 4.3. --- Detection of HBV-DNA in clinical serum samples --- p.148
Chapter 4.3.1. --- Crossectional study of patients with various categories of HBV related diseases --- p.148
Chapter 4.3.1.1. --- HBV-DNA detection in serum --- p.148
Chapter 4.3.1.2. --- Detection of HBV-DNA in peripheral blood mononuclear cells --- p.153
Chapter 4.3.2. --- Longitudinal studies of patients undergoing antiviral therapy --- p.159
Chapter 4.3.2.1. --- Serum HBV-DNA and HBeAg --- p.159
Chapter 4.3.2.2. --- HBV-DNA in peripheral blood leucocytes --- p.163
Conclusion --- p.166
Future perspectives --- p.168
References --- p.170