BACKGROUND: As the prevalence of metabolic risk factors in the American population has increased over time, so too has the diagnoses of non-alcoholic fatty liver disease (NAFLD). Within this spectrum of disease lies the potential for silent progression towards cirrhosis, leaving the patient with few options for treatment. Currently, the standard of care remains counseling on diet and exercise with the goal of reversing disease progression by addressing the underlying risk factors. LITERATURE REVIEW: Recent studies have shown that a correlation exists between low levels of serum 25-hydroxyvitamin D and hepatic injury from NAFLD. This has become an active area of research, due in part to the anti-inflammatory and immunoregulatory properties of vitamin D. The prospect of a simple and cost effective intervention that can exert its effects on the mechanisms behind the development of NAFLD is interesting and warrants further research. PROPOSED PROJECT: This proposal is for a double-blind, randomized, experimental study of vitamin D3 (cholecalciferol) versus placebo in a patient population of those with both clinically proven NAFLD and concomitant vitamin D deficiency. Liver fibrosis will be measured and staged with the use of FibroScan elastography. The statistical analysis thereafter will determine if a clinically significant reduction in hepatic fibrosis exists, compared with the results of the placebo group. CONCLUSIONS/SIGNIFICANCE: Should vitamin D prove to be an effective treatment option in reversing the progression of NAFLD, clinicians would be equipped with a simple and safe tool to augment their management of the patient. For those that experience barriers (i.e. lower socioeconomic status, other comorbidities, etc.) preventing them from improving diet and exercise, vitamin D would serve as an alternative therapy to aid in reducing their disease burden. Easier methods to treat their disease now projects improved quality of life years later.

Abstract In the search for more reliable blood markers for excessive alcohol consumption, considerable effort has been devoted to measurements of carbohydrate-deficient transferrin (CDT), which increases in body fluids as a result of prolonged alcohol intake. In the present work, three CDT methods, CDTect (Pharmacia & Upjohn), %CDT radioimmunoassay (%CDT RIA) by Axis (Oslo, Norway), and Axis %CDT turbidimetric immunoassay (%CDT TIA) were examined for their diagnostic performance in cases of alcohol abuse with or without liver disease. The diagnostic performance of CDT as a marker of alcohol abuse correlates positively with alcohol consumption. As compared with g-glutamyltransferase (GGT) and mean corpuscular volume of erythrocytes (MCV), which are conventionally used as laboratory markers of excessive ethanol consumption, CDT (CDTect) has the highest sensitivity (64%) at the specificity level of 100% in heavy drinkers consuming >100 g ethanol/day, but its sensitivity decreases to 34% in cases with an alcohol intake of <100 g/day, which hampers the use of CDT as a community screening method. Patients with alcoholic liver disease (ALD) have significantly higher CDT values than alcoholics with non-liver pathology. However, CDT is primarily increased in cases with an early stage of ALD, so that there is a weak negative correlation between CDT and disease severity, which may prove to be of diagnostic value. Especially in men, CDTect seems to achieve greater sensitivity than %CDT RIA or %CDT TIA for detecting recent alcohol abuse among heavy drinkers, but it does have a significant correlation with serum transferrin, especially in individuals reporting social drinking or no alcohol intake. This should be considered when interpreting the assay results in patients with increased serum transferrin. %CDT methods achieve greater specificity than CDTect when analyzing samples from patients with high serum transferrin concentrations. Acetaldehyde-protein adducts are formed in the body after excessive ethanol intake, and their formation triggers antibody production, which may contribute to some forms of tissue damage seen in alcohol abusers. To obtain more information on the association between serum antibodies against acetaldehyde adducts, ALD and alcohol consumption, assays for antibodies against albumin and haemoglobin adducts were performed. Antibodies of the immunoglobulin (Ig) isotypes A, G, and M against acetaldehyde-adducts are formed in patients with prolonged heavy alcohol consumption. IgA titres in ALD patients are significantly higher than those found in patients with non-alcoholic liver disease, non-drinking controls, or heavy drinkers with no signs of liver disease. Anti-adduct IgG titres, in turn, are increased both in ALD and in heavy drinkers with no signs of liver disease as compared with non-alcoholic liver disease patients or non-drinking controls. It appears that anti-adduct IgA, IgG and IgM titres in ALD patients correlate with the severity of the liver disease. Although this association is a limitation for the usefulness of these antibodies as markers of alcohol abuse, it may serve as a basis for the differential diagnosis of alcohol-induced liver disease.

Background: The number of patients dying with advanced liver disease is rising dramatically. However, little is known about the experiences of these patients and their families in respect of their care and everyday life with the disease. Palliative care services are traditionally focused on cancer and more recently on other types of organ failure, but liver disease is relatively neglected. Aim and objectives: This study aimed to broaden our understanding of the experience of living and dying with advanced liver disease. Specifically, it sought to explore the dynamic physical, psychosocial, existential and information needs of patients and their lay and professional carers, and to review their use of health, social and voluntary services. Additionally, this study examined the utility of a qualitative longitudinal, multi-perspective methodology in end-of-life research. Methods: This study employed qualitative, multi-perspective serial in-depth interviews. Patients with different aetiologies of liver disease were recruited in hospital. They and their lay carers were interviewed up to three times over one year. Single interviews were undertaken with case-linked professionals. Interviews were recorded, transcribed and analysed using grounded theory techniques and NVivo 9. Results: 15 patients, 11 lay carers and 11 professional carers were recruited, and 53 interviews conducted. Uncertainty was the key experience at all stages of the illness, across all domains, and for all participants: patients, lay carers and professionals. This uncertainty related to the nature of the illness, the unpredictability of disease pathway and prognosis, poor communication and information-sharing, and complexities of care. Coping strategies demonstrated a continuous quest to manage uncertainty. Current care arrangements were a poor fit with the high levels of physical and psychosocial need identified. The ubiquitous uncertainty meant that a care planning approach was difficult to introduce. Employing a qualitative longitudinal, multi-perspective approach emerged as a useful and effective way in which to conduct research with this patient group and contributed new learning with regard to its application in end-of-life research. Conclusion: This study identified uncertainty as the central pervasive factor in the experiences of patients, lay and professional carers. The needs of this patient group are currently poorly met from diagnosis to bereavement. Uncertainty makes advance care planning important, but difficult to know when to start. More needs to be done to ensure that people living and dying with advanced liver disease and their families benefit from appropriate and timely supportive and palliative care.

Obesity in adults and children is associated with increased cardiovascular mortality and morbidity. This is forecast to increase markedly in the next decade as childhood obesity is a burgeoning epidemic. Excess weight is clearly associated with insulin resistance, increased circulating triglycerides, and hypertension, all of these are related to progressive heart and liver disease. Ectopic fat deposition within organs is reported to cause lipotoxicity, which may lead to dysfunction and disease, but there have been few human studies to confirm this. This doctoral thesis set out to study the early pathophysiology of obesity in adults and children using in vivo magnetic resonance (MR) imaging and spectroscopy to assess the composition and function of the heart and liver in lean and obese individuals. The central tenet of this project was to establish and validate a clinically viable method for measuring the fat content of viscera safely and accurately, and to determine a normal range for the triglyceride content of the heart and liver. The initial study demonstrated that the heart remodels in response to weight loss, with over 20% reduction in LV mass, confirming that excess weight is genuinely a modifiable risk factor. Then, using spectroscopy, it was established that the healthy myocardium has a median triglyceride content of 0.37% (IQR 0.24% - 0.47%), which increases linearly in overweight and obese adults. Obesity, in the absence of any confounders, was also associated with a 10% reduction in cardiac contractile function. In comparison, healthy liver median lipid content was 0.67% (IQR 0.44% – 0.88%), which increased in obese adults to 2.9% (IQR 1.6% - 7.6%). There was a graded association between ectopic fat deposition in the liver and dyslipidaemia in adults, characterised by increased circulating triglycerides and reduced high-density lipoprotein. This dyslipidaemia may impair reverse cholesterol transport, and thus could be expected to exacerbate weight gain. Among obese and overweight subjects, there were some with severe steatosis and evidence of coexistent hepatic inflammation and fibrosis. To verify the accuracy of these spectroscopic measures for ectopic fat, a blinded, prospective comparison of non-invasive assessment of unselected liver disease in liver biopsy patients was completed. Liver disease presents with one or more of steatosis, fibrosis and haemosiderosis, all of which are associated with adverse cardiovascular outcomes. Fifty patients were recruited, and interobserver variability among pathologists was measured for histological reference standards for fat, fibrosis and iron deposition. MR measures of each of these metrics predicted the fibrosis, steatosis and haemosiderosis scores accurately. This enabled precise tissue characterisation of all forms of liver disease, including steatohepatitis, with one non-invasive test, to allow the diagnosis and monitoring of hepatic conditions. Lastly, all these new biomarkers of early cardiac and liver disease associated with excess weight were applied to obese and lean children, to understand whether ectopic fat played a substantial role in early life. Obese children had increased ectopic fat in their hearts and livers, as well as impaired strain, evidence of dyslipidaemia, and in some cases evidence of active steatohepatitis, comparable to adults with severe disease. The thesis therefore demonstrates that in vivo magnetic resonance techniques can be used for accurate measurement of visceral lipid content. Furthermore, there is evidence of significant ectopic fat deposition in both adults and children, with evidence of organ dysfunction, which raises the possibility that cardiovascular magnetic resonance may be of value to risk stratify obese individuals based on organ involvement. Finally, the developed methods may have broader applicability and offer a promising new method for the non-invasive diagnosis of chronic liver disease in other clinical settings.

archives@tulane.edu / Alcoholic and nonalcoholic fatty liver disease is projected to be the most common cause of liver disease in developing countries. The main significant risk factors are obesity, diabetes mellitus type 2, cardiovascular disease, and dyslipidemia. Louisiana is ranked seventh in liver cancer diagnoses and ranked sixth in the leading cause of death. Recent findings indicated that multifaceted stress response due to the accumulation of fatty acids from the diet is the driving force of disease progression. We sought to study multifaceted integrated stress response (ISR) in liver cells cultured with saturated fatty acids. Understanding the process that ISR takes to either induce or inhibit autophagy, self-eating machinery, in strongly permissive HUH 7.5 cells is vital when treating liver abnormalities. The major protein kinase, P-EIF2 alpha, was the targeted factor contributing the most to autophagy due to its functional link to the endoplasmic reticulum, mitochondria, and cellular membrane by further assessment using the inductive drug, Sephin 1. HUH, 7.5 liver cells are treated with increasing amounts of palmitic acid for 24 hours in DMEM with 10% FBS. ISR activated after substantial cellular damage leading to autophagy impairment. The cell culture was assessed for lipid accumulation, and the expression of PKR, IRE1 alpha, PERK, ATF6, P-EIF2 alpha, HRI, MTORC1, GCN2, P62, and LC3B was achieved by immunoblot analysis. Membrane fluidity PKR, lysosomal MTORC1, and protein synthesis GCN2 activated to elicit an integral response to the ISR pathway. Endoplasmic reticulum protein kinases induced in response to UPR activation lead to an integration of the P-EIF2 alpha pathway. Mitochondrial stress heme regulated inhibitor proliferated to provoke an activation in the significant protein kinase leading to autophagy impairment. The P-EIF2 alpha kinase invoked autophagic deficiency even when dephosphorylation was prevented by Sephin 1 drug treatment. ISR constrained autophagy in the liver-derived cell line due to the accumulation of the toxic saturated fatty acid. Keywords: palmitate, autophagy, fatty liver disease, integrated stress response, Sephin 1 / 1 / Glory Ogunyinka

Indiana University-Purdue University Indianapolis (IUPUI) / The ultimate goal of this study is to build a decision support system to aid the pathologists in diagnosing Non-Alcoholic Fatty Liver Disease (NAFLD) in both adults and children. The disease is caused by accumulation of excess fat in liver cells. It is prevalent in approximately 30% of the general population in United States, Europe and Asian countries. The growing prevalence of the disease is directly related to the obesity epidemic in developed countries. We built computational methods to detect and quantify the histological features of a liver biopsy which aid in staging and phenotyping NAFLD. Image processing and supervised machine learning techniques are predominantly used to develop a robust and reliable system. The contributions of this study include development of a rich web interface for acquiring annotated data from expert pathologists, identifying and quantifying macrosteatosis in rodent liver biopsies as well as lobular inflammation and portal inflammation in human liver biopsies. Our work on detection of macrosteatosis in mouse liver shows 94.2% precision and 95% sensitivity. The model developed for lobular inflammation detection performs with precision and sensitivity of 79.3% and 81.3% respectively. We also present the first study on portal inflammation identification with 82.1% precision and 88.3% sensitivity. The thesis also presents results obtained for correlation between model computed scores for each of these lesions and expert pathologists' grades.

Student Number : 0101826W - M Med dissertation - School of Clinical Medicine - Faculty of Health Sciences / Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with hepatic histology that resembles alcoholic liver disease. It is a frequent cause of chronic liver disease and is attracting increasing scientific attention worldwide. I explored the possibility that increased gastrointestinal alcohol production may have a role as a “second hit” in the pathogenesis of NAFLD in study subjects with the metabolic syndrome. In an attempt to investigate this hypothesis, this study looked at blood, urine and breath levels of alcohol in patients with the metabolic syndrome versus matched age and ethnic group healthy controls. Of the twenty study subjects, 80% had dyslipidaemia, 60% had hypertension and 70% had type 2 diabetes mellitus. Their mean BMI was 35.1±8.2 kg/m² (mean ± SD, P < 0.0001 versus controls). The serum aminotransferases were significantly elevated in the study subjects, their ALT levels being 57.4±44.79 U/L versus 17.4±4.60 U/L in the controls (95% CI 18.02 – 61.42, P < 0.001), and their AST levels 52.5±36.21 U/L versus 23.4±4.86 U/L in the controls (95% CI 11.99 – 46.20, P < 0.01). Seventy five percent of the study group had sonar features suggestive of fatty liver disease. Two adipocytokines, adiponectin and leptin, mediators of insulin resistance, an important factor in the development and progression of NAFLD, were also measured. Adiponectin levels were significantly lower (6875 ng/L versus 15475 ng/L; median value, P < 0.01), and leptin concentration levels significantly higher (13.56 ng/L versus 3.05 ng/L; median value, P < 0.05) in the study subjects than in the control group. Alcohol was detected in 60% of the study subjects, of which 35% tested positive for ethanol, 55% tested positive for methanol, and 30% tested positive for both ethanol and methanol. This was a statistically significant result, as none of the control group tested positive for any of the alcohols. The ethanol concentration in the study subjects’ blood was 7.14±3.28 mg% (mean ± SD), in their urine 3.71± 12.87 mg% (mean ± SD) whilst none was detected in their breath. The methanol concentration in the study subjects’ blood was 16.17±17.95 mg% (mean ± SD), in their urine 6.8± 13.58 mg% (mean ± SD) while their breath level was 2.05±3.19 mg (mean ± SD). This study therefore suggests that endogenous alcohol production may be indeed be involved in the pathogenesis of NAFLD in subjects with the metabolic syndrome. Not only ethanol but also methanol was detected in the subjects tested. Endogenous alcohol may therefore be responsible for the ‘second hit’ theory in the pathogenesis of NAFLD, and it is likely that formaldehyde, the metabolite of methanol may be a more potent toxin of hepatocyte injury as opposed to acetaldehyde, the metabolite of ethanol. The most likely source of the alcohol is from intestinal bacterial flora. These findings provide further insight into the pathogenesis of NALFD, suggesting other therapeutic alternatives such as the use of antibiotics and probiotics as a potential treatment strategy for NAFLD.

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The liver is a multi-function organ that plays important roles in nutrient metabolism, biochemical transformations and blood detoxification. The purpose of the current work was to optimize Blood Oxygen Level Dependent (BOLD) liver functional MR imaging and analysis to allow the distinction between healthy volunteers and subjects with chronic liver disorders known to lead to fibrosis and reduced liver function (in this case, Hepatitis-C). Liver BOLD signal can be modulated by breathing 100% 0 2 or through intake of a meal. Previous results using these stimuli have been inconclusive when comparing healthy and diseased livers. In addition, liver BOLD analysis has been traditionally carried out using general linear models (GLM). Since the liver has a dual blood supply (portal and arterial derived), its resultant haemodynamic response is complex. This makes it too difficult to employ GLM approaches, as they require the prediction and modeling of a response function. We chose a model-free, or data-driven approach, called principle component analysis (PCA) to analyze liver data. Initial optimization was done by determining the time of maximal hepatic portal vein (HPV) blood flow following ingestion of a controlled meal (235 mL of Ensure Plus®). Statistically significant increases in HPV flow resulted at all measurement intervals, with the maximal postprandial change (71% increase in comparison to the baseline flow) at thirty minutes after ingestion. Implementing acquisition and analysis optimizations with our dual liver challenge model (hyperoxia cycling in pre- and postprandial states), the PCA approach was able to detect all of the diseased livers (n=6), while missing four of the healthy subjects (n=ll). The GLM technique, on the other hand, did not detect two of the patients and two of the healthy subjects. Thus, if this liver challenge is to be used as a screening tool, a model-free data analysis approach is suggested as more appropriate since it minimizes the chances of reporting false-negative results (based on this preliminary cohort). Although more false positives were detected with this method, it is of less concern seeing as these inaccuracies can be screened using simple blood tests. Promising results were obtained in this project, however, further studies using data-driven approaches such as partial least squares (PLS) are needed. / Thesis / Master of Science (MSc)