Medicine (RMH) - Theses
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ItemHIV in Victoria's African communities: reducing risks and improving care( 2013)The acquired immunodeficiency syndrome caused by the human immunodeficiency virus is an important issue for Australia’s African communities. As in other industrialised countries, African immigrants are over-represented in Australia’s HIV epidemic, diagnosed late and endure social isolation after diagnosis, but focused responses, applied without understanding local HIV epidemiology and social context, risk intensifying stigma against African communities and African Australian people living with HIV in Australia. This study explored the social epidemiology and clinical features of HIV in Victoria’s African communities, collecting data from national and Victorian HIV surveillance databases, a clinical case series of African-born HIV patients and a qualitative inquiry with several African communities. Diverse geographical, biological, psychosocial and structural factors influenced exposure, diagnosis, clinical features and experience of living with HIV. Most exposure occurred in Africa, prior to migration, through heterosexual sex. Some occurred after migration, in Australia and abroad, through heterosexual sex and sex between men. Low self-perceived risk and lack of awareness of HIV in Australia contributed to exposure and delayed diagnosis. HIV was understood as a deadly, highly contagious “African” disease, posing little threat in Australia, being one of several intersecting challenges to the wellbeing and cohesion of African communities during resettlement. Understanding of HIV was based largely on experience in Africa and the process of HIV screening during immigration. HIV-related stigma, based on risk stereotypes of sexual immorality and fear of contagion and death, was the major barrier to social support and information. Key clinical issues for African-born PLHIV included high prevalence of TB and viral hepatitis. HIV treatment uptake was high and response was good. HIV exposure via sex between men led to HIV-1 subtype B infection; those with heterosexual or other exposure carried various non-B subtypes. African communities actively participated in the study leading to greater engagement in Victorian and national HIV responses. Study results provided insights into HIV epidemiology and clinical features in Victoria’s African communities and informed a conceptual framework that should further the understanding of HIV epidemiology in mobile and marginalised populations.
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ItemNovel markers in chronic liver disease and hepatocellular carcinoma( 2013)Chronic liver disease is associated with a number of metabolic changes and nutritional deficiencies, and these may have important consequences on the natural history of liver disease. Chronic liver disease is a continuum of progressive liver fibrosis with replacement of injured tissue by a collagenous scar. Fibrosis is a multi-step process and progresses at variable rates depending on the cause of the liver disease, environmental factors, and host factors. Cirrhosis is an advanced stage of liver fibrosis, and is a common cause of morbidity and mortality worldwide, including a predisposition to hepatocellular carcinoma of 2–7% per year. Hepatocellular carcinoma is one of the most common malignant tumours in the world accounting for 500,000 deaths per year, making it the 3rd most common cause of cancer death worldwide. In this thesis, I have reviewed the epidemiology, natural history and current investigations used in chronic liver disease and hepatocellular carcinoma. The progression of liver disease to cirrhosis, and hepatocellular carcinoma, are multi-step pathways. The poor outcomes highlight the need for novel factors to be explored to determine if they can predict and/or alter the natural history. In this thesis, I review the previous literature on CD163, CD147 and 25(OH)D as novel markers in liver disease assessment, to determine their relationship to liver disease severity and outcome. CD163 is a marker of activated macrophages and sCD163 is found in normal plasma. Upregulation of sCD163 occurs during inflammation and increased levels are seen in acute and chronic liver disease. Routine biochemistry and imaging are often insensitive for complications of chronic liver disease, and in this thesis we demonstrate that sCD163 levels predict chronic liver disease progression and portal hypertension. sCD163 is a good marker of chronic liver disease patients who have developed cirrhosis, stratifying patients based on the degree of liver impairment. High sCD163 levels may be used for screening purposes and in the early identification of the need for interventions, such as starting anti-viral treatment in patients with chronic viral hepatitis, or in screening for oesophageal varices. CD147 is a cell surface transmembrane protein that regulates tumour-stromal interactions, and induces the secretion of MMP in the tumor local environment, thus promoting tumor invasion and metastasis. CD147 is highly expressed on hepatocellular carcinoma cells and is associated with a poor prognosis. Soluble CD147 is released by shedding, and in this thesis, we demonstrate that increased sCD147 levels predict patients with more advanced hepatocellular carcinoma and a high likelihood of death within 90 days. This may assist in the identification of HCC patients with a poor prognosis and help clinician’s define patients unsuitable for ongoing active treatment. 1,25-dihydroxyvitamin D3 has been shown to exert potent immunoregulatory effects that are mediated through binding to an intracellular receptor protein, the vitamin D receptor. In this thesis, we demonstrate that vitamin D deficiency is more common in chronic liver disease and hepatocellular carcinoma compared to the Australian population. Higher serum 25(OH)D concentrations were demonstrated to be associated with a reduced risk of disease progression and death due to liver disease in both the chronic liver disease and hepatocellular carcinoma cohorts. Vitamin D deficiency is a common problem in the Australian population.
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ItemCardiovascular disease in systemic sclerosis: an emerging association?( 2013)Systemic sclerosis (SSc) is a multiorgan connective tissue disease in which vasculopathy features prominently. Whilst microvascular disease occurs universally, it is not yet established whether the prevalence of macrovascular disease is increased. This thesis addresses several aspects of cardiovascular disease in SSc. In the first study, survival and predictors of mortality were examined in a nation-wide Australian cohort of patients with connective tissue disease-associated pulmonary arterial hypertension (PAH). Three-year survival was found to be 73% and an independent survival benefit was observed with both anti-coagulation with warfarin and combination pulmonary vasodilator therapy. In the second study, a cross-sectional analysis of the prevalence of coronary heart disease (CHD) in a nation-wide Australian cohort of patients with SSc was performed. CHD was found to be more prevalent than in controls drawn from two contemporaneous population-based studies, with a fully adjusted odds ratio of 3.2 (95% CI 2.3 to 4.5) for CHD in SSc patients compared with controls. The metabolic conditions hypercholesterolaemia, diabetes mellitus and obesity, however, were all less prevalent in SSc patients than in controls. Furthermore, within the SSc cohort CHD was independently associated with PAH. In the third study, a cross-sectional analysis of arterial stiffness in SSc patients with comparison to age- and sex-matched controls was performed. Augmentation index (AIx) was higher in SSc patients than in controls (31.0% [IQR 25.7 – 38.7] vs 23.8% [IQR 13.5 – 30.1], p < 0.001), but pulse wave velocity was not. Within the SSc cohort higher AIx was associated with calcium channel blocker (CCB) use, a paradoxical finding that could reflect the impact of microvascular disease in these patients given that CCBs are first-line therapy for Raynaud’s phenomenon. Finally, a study of primary myocardial disease using tissue Doppler imaging and the serum biomarkers high-sensitivity troponin T (hsTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was undertaken in SSc patients free from CHD or reduced left ventricular (LV) ejection fraction. NT-proBNP, but not hsTnT, was found to be higher in SSc patients with systolic dysfunction defined as LV long-axis or right ventricular long- or short-axis systolic dysfunction. hsTnT was independently associated with LV mass index, LV long-axis systolic dysfunction and lack of anti-centromere antibody; whilst NT-proBNP was independently associated with E/e`, a marker of left atrial pressure. The studies described in this thesis contribute to the growing body of data suggesting that the prevalence of macrovascular disease is increased in SSc. Uncertainty remains, however, as to the exact nature of CHD in SSc, and as to the relationship between microvascular disease and macrovascular disease. These issues, as well as the relationship of CHD with primary myocardial disease and PAH in SSc warrant further exploration in future studies.
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ItemFunctional analysis of sodium channel gene variation in epilepsy( 2013)A genetic etiology of epilepsy is widely accepted in 50-70% of all epilepsy syndromes. With genome sequencing now increasingly efficient and affordable, more and more novel genes and mutations are being discovered that are associated with epilepsy. However, most of the mutations have been discovered in genes that code for ion channels which has led to the theory that the genetic epilepsies are a family of channelopathies. The voltage-gated sodium channel family have been particularly implicated with over 800 variants discovered in this gene family. Given their critical role in regulating neuronal excitability it is not surprising that genetic variations in sodium channels can have functional and potentially devastating consequences. With a focus on the voltage-gated sodium channels, the three chapters in this thesis used high-throughput automated planar patch-clamp technology to try and develop a deeper understanding of genetic risk in epilepsy. Chapter two examines a novel cause in a mouse model of absence epilepsy that harbours a mutation in the Scn8a gene. The phenotype of this mouse is enhanced on the C3H background, as opposed to C57, where the C3H animal also has a mutation in the Scn2a gene. The individual biophysical profiles of these two mutations were examined on the Nanion patchliner, and their potential genetic interaction was investigated in a computer model of a layer 5 pyramidal neuron, to see if this could be explained by a biological interaction at the axon initial segment. The results revealed an overall loss of function of the NaV1.6V752F mutant, and an overall gain of function in the NaV1.2V929F mutant. When these changes were implemented in the computer model, it revealed that the output was dominated by the NaV1.2V929F mutant, which suggests there is not a biological interaction of these two genes at the axon initial segment. Alternative scenarios where there may be an alternative site for biological epistasis will be revealed with future studies using immunohistochemistry and brain slice patch clamp recording in the mice. It may also be the case that the NaV1.2V929F mutant is not a modifier of the NaV1.6V752F mutant, which will be revealed by genetic studies to identify the modifier genes. The third chapter examined the modulation of NaV1.2 and NaV1.1 by the β1 auxiliary subunit. As mutations in the β1-subunit have been detected in patients with epilepsy, understanding their impact on subunits from excitatory and inhibitory neurons is critical for understanding how this variation impacts on risk for epilepsy. There was a differential modulation revealed where β1 had a greater functional effect on the NaV1.2 channel but a greater effect on current density on the NaV1.1 channel. Therefore if a variant in β1 experiences a functional change this suggests differentially altered levels of excitation and inhibition in the brain, which could feasibly result in an epileptic phenotype. The fourth chapter looked at exploiting the high-throughput capabilities of the Nanion patchliner, and examined eight mutations in the β1-subunit co-expressed with NaV1.1 and NaV1.2 that have been associated with epilepsy. With this influx of data we needed to devise a new way to represent this data, and converted all raw measurements to effect size values, and represented them on tornado diagrams. With this measurement we could then more easily directly compare parameters from the individual protocols and calculate averages both across mutations, and across parameters. From this data set it is quite apparent that the β1 mutants modulate the α-subunits quite differently, both comparing α-subunits, and comparing mutations. More importantly however this chapter highlighted a new way of thinking about analysis of high-throughput electrophysiology data. As people continue to look into the genetics of epilepsy and reveal novel genes and novel mutations implicated in the disease, we need to look for new ways to tame the genetic complexity, and look for points of convergence. High-throughput technology allows us to decrease the time lapse between the discovery of the genetic variants and the corresponding functional analysis. And the type of analysis as suggested in chapter four, enables us to start to look for points of convergence in the functional data. This data can then be used to train clustering algorithms to group the variants based on their ‘channelomic’ profile. To do this we need a large volume of functional data obtained from variants that have strong corresponding phenotypic data, and future studies should endeavour to accomplish this.
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ItemDetecting new-onset diabetes and the effect of prednisolone on glycaemia after renal transplantation( 2013)Background: A significant impediment to long-term graft survival after kidney transplantation has been the morbidity and mortality attributable to cardiovascular disease. New-onset diabetes after transplantation (NODAT) is an independent predictor of cardiovascular events and prednisolone immunosuppression is a major modifiable risk factor for NODAT. Aims: This study aimed to investigate the relationship between prednisolone and post-transplant glycaemia and to identify strategies to reduce the incidence and improve the detection of NODAT. Methods: In kidney transplant recipients 3-4 weeks post-transplant, the accuracy of limited sampling strategies for assessing exposure to biologically active free prednisolone was tested and the relationship between free prednisolone exposure and glycaemia was determined using continuous glucose monitoring (CGM). CGM was also used to compare the glycaemic effects of once daily and divided twice-daily prednisolone dosing. Finally, the sensitivities of screening tests for NODAT were compared at various time points during the first year after kidney transplantation. Results: Free prednisolone exposure was accurately predicted using limited sampling strategies that incorporated two concentration samples within four hours of dosing. There was significant inter-individual variability in free prednisolone exposure but minimal intra-individual variability, and no association between bodyweight and free prednisolone exposure was observed. Free prednisolone exposure correlated with hyperglycaemia, measured by peak glucose and exposure to glucose concentrations of 7.8mmol/L or greater. Dividing the total daily prednisolone dose into two equal doses 12 hours apart reduced hyperglycaemia and glycaemic variability. Once daily prednisolone administration resulted in a peak in plasma glucose 7-8 hours later and during the initial post-transplant hospitalisation 70% of patients without pre-existing diabetes developed diabetic range blood glucose levels. With moderate prednisolone doses six weeks post-transplant, 4pm blood glucose assessment was more sensitive than the oral glucose tolerance test in detecting NODAT. With low maintenance prednisolone doses from three months onwards, glycated haemoglobin (HbA1c) had comparable sensitivity to the oral glucose tolerance test in detecting NODAT. Fasting plasma glucose testing was inadequate for detecting NODAT at all stages in the first year post-transplantation. Conclusions: After kidney transplantation free prednisolone exposure correlates with glycaemia and divided twice-daily dosing reduces hyperglycaemia. Therefore therapeutic drug monitoring and divided daily dosing may reduce the incidence of NODAT. With morning prednisolone, hyperglycaemia is best detected by afternoon glucose testing early post-transplant, and either HbA1c or OGTT from three months post-transplant.
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ItemAtrial fibrillation and heart failure: the impact of atrial fibrillation on cardiac remodeling( 2013)Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular conditions that individually are associated with significant morbidity and mortality. Each has a tendency to promote the other, with grave outcomes resulting when they occur in combination. Despite the prevalence of concurrent AF-HF, pathophysiologic interactions between the two conditions remain to be well understood due in part to their complex and dynamic nature. Utilizing a wide range of complementary laboratory methods in the experimental setting, as well as start-of-the-art imaging and electrophysiologic techniques in a series of clinic studies, this thesis explores the relationship between AF and HF, focusing in particular on the influence of AF on left ventricular remodeling. Two highly novel findings include the following: (i) the irregular ventricular rhythm associated with AF has an adverse impact on ventricular calcium handling and contractile function independent of rate-related effects, and (ii) diffuse ventricular fibrosis resulting from tachycardia-mediated cardiomyopathy persists long after resolution of tachycardia and appears to correlate with AF burden, suggesting that fibrosis may be cumulative with successive periods of poor rate control. These results imply that a strategy of rate-control alone in the management of combined AF-HF is insufficient to protect the left ventricle from the adverse influence of AF, and add weight to the argument for early rhythm control. Further major findings relating to clinical management of combined AF-HF include the following: (i) the pattern of ventricular fibrosis as assessed by cardiac magnetic resonance (CMR) imaging is useful in selecting patients most likely to recovery LV function following catheter-based therapy for AF, and (ii) post-contrast CMR T1 mapping identifies changes in atrial myocardial tissue that parallel electrophysiologic remodeling and are suggestive of atrial fibrosis. Thus, CMR imaging may have a key role in individualizing therapy for patients with combined AF-HF, and place its routine use in clinical practice on the horizon.
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ItemGlioma stem cells: novel signalling pathways and potential therapeutic advances( 2013)High grade glioma continues to portend a dismal prognosis for patients, despite ongoing, albeit incremental, advances in surgery, irradiation and chemotherapy. This has necessitated an overhaul in the understanding of disease pathogenesis, and the search for a superior model for screening of small molecule therapeutic agents, especially in light of the poor translational outcomes generated from work on traditional, serum-fed, glioma cell lines. The cancer stem cell hypothesis has revolutionised neuro-oncological research, and the discovery of the glioma stem cell (and in particular, its characteristic in vitro gliomasphere-producing qualities) has led to new avenues of thinking, in terms of glioma proliferation, invasiveness and mechanisms of chemo- and radioresistance. Coupled with this has been further characterisation of the genetic constitution of high grade gliomas, which show Pi3k-related aberrations in up to 90% of such lesions, related mainly to EGFR amplification, EGFRvIII expression and PTEN mutation. This study has led to the production of sixteen gliomasphere-producing (or glioma stem cell) clones, from the collection of sixty-six individual specimens, which included lower grade lesions. It was confirmed that this set of stem cell lines exhibited all of the trademark characteristics of a tumour stem cell, characterised by a potential for self-renewal, pluripotency, proliferation, and tumour formation upon xenograft transplantation. Western blot analysis confirmed that thirteen of the sixteen reproducible stem cell clones demonstrated hyperphosphorylated Akt. This led to an investigation of suitable Pi3k inhibitors via a series of in vitro lactate dehydrogenase cytotoxicity assays, which showed that the N10-substituted phenoxazine, Akt inhibitor Akt X, was the only agent which demonstrated effective in vitro cytotoxic potential against glioma stem cells. Interestingly, Akt X was shown to be more efficacious against those glioma stem cell clones which failed to express PTEN protein on Western blot analysis, and this result was found to be statistically significant. Further in vitro LDH cytotoxicity assays revealed glioma stem cells were largely resistant to conventional irradiation and temozolomide, but this was reversed by Akt X, confirming this agent’s potential as a successful stem cell-specific agent in the clinical management of high grade glioma patients. Extensive exome sequencing analysis confirmed the presence of a number of genetic variants in the glioma stem cell clones, whose putative effect and expression concerns signalling pathways not commonly reported in conjunction with gliomagenesis in the past, including the WNT/-catenin and SWH pathways. Western blot analysis and immunohistochemistry demonstrated that the glioma stem cell clones overexpress YAP, a protein whose activation is normally suppressed via the intact SWH pathway, identifying it as a prospective, glioma stem cell-specific oncoprotein, and target for future development in pharmacotherapeutics.
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ItemCharacterisation of the nuclear pore abnormalities in the intestinal zebrafish mutant, flotte lotte (flo)( 2012)The evolution of eukaryotic cells is defined by the compartmentalisation of the genetic material inside the nucleus, segregated from cytoplasm by a nuclear envelope. This barrier is punctuated by approximately 3000 large multi-protein structures known as nuclear pore complexes, which permit the bidirectional transport of protein and RNA molecules between the nucleus and cytoplasm. This study provided the opportunity to investigate the importance of the nuclear pore protein Elys during vertebrate development. Zebrafish mutants generated by ethylnitrosourea (ENU) mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes. Our interest has focused on a panel of ENU generated mutants exhibiting a variety of defects in the formation and differentiation of the intestinal epithelium. One of these mutants, flotte lotte (flo), harbours a premature stop codon in the coding sequence of the nuclear pore component elys (embryonic large molecule derived from yolk sac). Elys is an essential component of the nuclear pore complex, yet surprisingly, its mutation in the flo mutant does not result in a global dysfunction in nuclear pore formation throughout the developing zebrafish embryo. Instead, flo mutants exhibit tissue-specific abnormalities in the development of the intestinal epithelium, liver, pancreas and eye; organs that are highly proliferative from 48hpf. We show that this time-point coincides with the exhaustion of maternally-deposited stocks of elys mRNA from flo embryos. Not surprisingly, we found that the ensuing inability to create new nuclear pore complexes appears to impact most severely on these rapidly proliferating tissues. Using multi-photon microscopy we reconstructed three-dimensional renditions of the endodermal organs in wild-type and flo larvae. Compared to the highly elaborated and polarized intestinal epithelium of wild-type zebrafish, the intestinal epithelium in flo is thin, unfolded and poorly polarised. Moreover, nuclear pore complexes in flo intestinal cells are not embedded in the nuclear envelope but are found in profuse cytoplasmic aggregates. Catastrophic levels of apoptosis accompany the loss of a functional nuclear envelope in intestinal epithelial cells. Thus, flo mutants provide an opportunity to identify signals that commit nuclear pore-deficient cells to an apoptotic fate. We found that the apoptotic response observed in the flo intestine is mediated via a Tp53-independent mechanism. Since Elys function is critical for the integrity of proliferative cells in zebrafish, we investigated whether ELYS is also critical for the proliferation of human cancer cells. We discovered a strong up-regulation of ELYS expression in many cancers when compared to their respective normal tissues. We observed ELYS to be ranked in the top 1% of all up-regulated genes investigated in gene expression studies of colorectal, kidney, liver and breast cancers available in the Oncomine database. The discovery that ELYS is frequently over-expressed in human colorectal cancer suggests that our functional genomics approach to novel cancer gene discovery using zebrafish mutants is valid. Moreover, we propose that targeted approaches to disabling ELYS synthesis or function may activate apoptosis in colorectal cancer cells and provide a useful therapeutic approach in the future.
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ItemAnalysis of erythropoiesis through ENU mutagenesis( 2013)Abnormalities in erythrocytes, the cells essential for normal respiratory exchange throughout the body, are amongst the most common disorders worldwide. The regulation of erythropoiesis is incredibly complex and much of our current understanding of the mechanisms underpinning this regulation has come from extensive study on the inherited forms of red cell abnormalities in humans and mice. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has been highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel genes or alleles regulating erythropoiesis and to establish new mouse models of human red cell disorders, we have undertaken a dominant ENU mutagenesis screen in mice. This thesis details the study of four ENU red blood cell (RBC) mouse mutants that have provided powerful insights into the genes involved in erythropoiesis. β-thalassemia is one of the commonest monogenic disorders worldwide, characterised by serious morbidity and early mortality. Almost all mouse models of β-thalassemia have been generated by the targeted deletion of the adult β-globin genes that, whilst recapitulating the clinical phenotypes of human β-thalassemia, do not reproduce the genetic mechanisms of human disease. Using ENU mutagenesis, we have identified two red cell mutants, RBC13 and RBC14, obtained from independent ENU mutagenesis screens that are the first mouse models to accurately recapitulate human β-thalassemia at the genomic level. This study highlights the effectiveness of ENU mutagenesis screens in establishing novel mouse models relevant to human disease. The major determinants of red cell volume regulation are the potassium-chloride (K-Cl) cotransporters, KCC1 and KCC3, mediating the transport of K+ and Cl- out of cells. The molecular mechanisms responsible for KCC1 and KCC3 regulation have only recently been meaningfully studied. Previous studies on KCC3 described the regulatory phosphorylation of two specific threonine sites in the C-terminal cytoplasmic domain, demonstrated to mediate K-Cl cotransport inhibition. The ENU mutagenesis screen described in this thesis has identified a red cell mutant (RBC10) which constitutes the first known activating mutation of KCC1, and defines a critical residue in KCC1 that regulates red cell volume. Our results demonstrate that the RBC10 mutation inhibits phosphorylation of threonine residues homologous to that described in KCC3. Further, the RBC10 mutant will be informative in the study of the involvement of KCC1 in the human disorders sickle cell disease and hereditary stomatocytosis. Disorders of iron are of global health concern. Mouse models have played a significant role in elucidating the genes and molecular mechanisms underpinning iron metabolism. Dynamin 2 (DNM2) is an essential component of clathrin-mediated endocytosis. Mutations of DNM2 are highly disease-specific and have been implicated in three forms of human disease, centronuclear myopathy, Charcot-Marie Tooth neuropathy, and more recently Tcell leukemia, but no recognised red cell abnormalities. ENU mutagenesis has identified a red cell mutant (RBC12) containing a novel DNM2 mutation in the GTPase domain, which perturbs GTPase activity. Our studies on the RBC12 mice provide the first in vivo evidence of the involvement of DNM2 in red cell iron uptake and possible implications of DNM2 involvement in human iron deficiency anemia.
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ItemIdentification of predictors of epilepsy outcomes( 2013)BACKGROUND: Epilepsy is a group of complex conditions characterized by the occurrence of recurrent spontaneous seizures, associated with high risk of brain abnormalities, co-morbid disorders and premature mortality. These issues in patients with new onset-seizures have not been well characterized. Furthermore, the first anti-epileptic drug (AED) treatment will fail in more than 50% of patients because of inadequate seizure control or intolerable adverse effects. Substitution to a newer AED may result in better outcomes. AIMS: (1) To determine the frequency and type of potentially epileptogenic abnormalities identified on MRI, in patients presenting with a possible new-onset seizure disorder, and their association with abnormal electrical activity on electroencephalogram (EEG), the frequency of co-morbidities and injuries, and the incidence of mortality (2) To examine whether patients who fail their first AED will have better neuropsychiatric, neurocognitive, quality of life (QOL), bone density and content, body composition and metabolism outcomes if substituted to monotherapy treatment with a newer-generation AED, levetiracetam, compared with another older-generation AED, valproate or carbamazepine. METHODS: (1) A consecutive series of 993 patients (597 males [61%], mean (SD) age: 42.2 (18.8) years, range: 14.3-94.3 years) who presented to an adult First Seizure Clinic over a ten year period were studied. The MRI scans were acquired using a dedicated epilepsy protocol or other specific protocols depending on clinical indication on either a 1.5T or 3.0T scanners. The co-morbidities and injuries were obtained from two state-wide administrative datasets, the Victorian Admitted Episodes Dataset (VAED) and the Victorian Emergency Minimum Dataset (VEMD), for the period from June 18, 1999 to June 30, 2010. Mortality data were collated from the National Death Index (NDI) developed by the Australian Institute of Health and Welfare (AIHW). (2) A randomized comparative trial was undertaken. Participants with focal epilepsy who had failed monotherapy with phenytoin sodium, carbamazepine, or valproate sodium were randomized to substitution monotherapy with levetiracetam (n= 51) or a different older AED (n= 48). Assessments were performed at baseline, 3 months, and 12 months using questionnaires measuring neuropsychiatric, QOL, seizure control, AED adverse effects, and neurocognitive outcomes. Assessments of bone density and content, body composition and metabolism were performed at 3- and 15 months after randomization. The assessments included: areal bone mineral density (aBMD) at lumbar spine, total hip, forearm, and femoral neck and total body bone mineral content [dual energy x-ray absorptiometry (DXA); Hologic QDR® 4500A densitometer], body composition [Hologic Software Version 5.73], peripheral quantitative computed tomography at non-dominant radius and tibia [pQCT; Stratec XCT 3000], serum markers of bone turnover, sex and metabolism hormones, questionnaires for bone health and lifestyle, blood pressure and anthropometry. RESULTS: (1) Potentially-epileptogenic lesions were detected in 177 (23%) of the patients in the First Seizure Clinic Cohort. Their frequency was higher in patients who had focal-onset seizures (53%). MRI and EEG were concordant in terms of the presence or absence of epilepsy associated abnormality in 62%, with 18% having an abnormality on both utilities. Almost 65% of patients with epilepsy had, at least, one co-morbid disorder as defined by an epilepsy-specific co-morbidity index, and 32% presented to Emergency Departments after sustaining an injury. Those patients were more likely to be males, have lesional focal epilepsy, frequent seizures and be diagnosed with epilepsy at 65+ years of age. The overall case mortality was 8.3% (82 cases) in the cohort. Standardized mortality ratio (SMR) for patients with new-onset seizures was 2.2 (95% CI 1.73-2.80); p< 0.001. The highest excess mortality was seen in younger patients (< 60 years). Proportionate mortality ratio (PMR) was highest for deaths from cerebrovascular diseases (16%) and malignant neoplasms (16%). Epilepsy was listed as the leading cause of death in 10%. (2) In the RCT, there were no differences in depression scores at 3 months between the treatment groups (improvement in 39.5% of the levetiracetam group vs. 34.1% of the older AED group; p= 0.60), but a greater proportion of the older AED group improved on the 89-item Quality of Life in Epilepsy Inventory (QOLIE-89) compared with the levetiracetam group (71.1% vs. 48.8% respectively; p= 0.04). The QOL, anxiety, and AED adverse effects scores were improved in both groups at 3 and 12 months after randomization. There were significant decreases in both treatment groups in aBMD at the lumbar spine (-9.0%; p< 0.001 in the levetiracetam group and -9.8%; p <0.001 in the older AED group), forearm (-1.46%; p< 0.001 and -0.96%; p< 0.001 respectively) and femoral neck (-0.47%; p= 0.026 and -1.45%; p< 0.001 respectively) on DXA scanning. The total hip aBMD significantly decreased in the older AED group (-0.84%; p< 0.001). The treatment groups did differ only in the change in femoral neck aBMD (p= 0.005). The serum levels of C-terminal telopeptides of type I collagen (βCTX), a marker of bone resorption, decreased in both groups (-16.1%; p= 0.021and -15.2%; p= 0.028 respectively) while procollagen 1 N-terminal peptide (P1NP), a marker of bone formation, significantly decreased in the older AED group (-27.3%; p= 0.008). There was no significant change in percentage abdominal fat in either treatment group. CONCLUSIONS: (1) MRI reveals potentially-epileptogenic lesions in a minority of patients who present following a newly-diagnosed seizure disorder. Lesions are most common in patients who have experienced focal seizures. The presence of a potentially-epileptogenic MRI lesion did not influence the likelihood of having an abnormal EEG. The majority of patients with new-onset seizures who consult a specialized First Seizure Clinic suffer from somatic or psychiatric co-morbid disorders. This finding should provide useful implications in the diagnosis and management of epilepsy and co-existing conditions, as well as in health care provision. Patients with new-onset seizures have a two-fold increased mortality compared with the general population. Cerebrovascular disease, and neoplasms, but not epilepsy, were the leading causes of death in epilepsy. (2) Substitution monotherapy in a patient who is experiencing ongoing seizures or tolerability issues is associated with sustained improvements in measures of QOL, psychiatric and adverse events outcomes. The significant bone loss at the lumbar spine, forearm, and femoral neck seen in both treatment groups and the modest reduction in bone turnover indicates that the adverse effects on bone health of chronic AEDs is likely occur with the newer-generation AEDs as well the older-generation AEDs.