A 72 year-old-patient without known past medical history presented to the hospital with worsening cough, dyspnea on exertion, decreased appetite, weight loss for two months. Prior to admission, he was treated with a 10- day course of levofloxacin and prednisone as a case of bronchitis with minimal improvement. Then he started to develop red urine with marked changes in mental status. On physical examination, the patient had notifiable scleral icterus, confusion and abdominal tenderness in the right upper quadrant. On admission his labs were significant for alkaline phosphatase 541, aspartate transaminase 557, alanine transaminase 94, total bilirubin 8.6, lactate 11.7. CT scan of abdomen showed hepatosplenomegaly, mild ascites and trace bilateral pleural effusion. Work up with Viral hepatitis serology, cryptococcal antigen, histoplasma antigen, respiratory virus panel, Epstein Barr virus tests were negative. Anti-nuclear antibodies (ANA) and anti-mitochondrial antibody were also negative. Blood level of amylase, lipase, acetaminophen and alcohol were negative at admission too. The patient was started initially on broad spectrum antibiotics, N-acetyl cysteine empirically and aggressive intravenous fluid hydration. Patient condition rapidly worsened and he developed profound shock requiring mechanical ventilation and started on stress dose steroid and pressor support. Upon further investigation, patient was noted to take terbinafine for toe onychomycosis (day 112). Ferritin level was elevated to 1596 with 93% iron saturation. Ceruloplasmin level was normal. Patient was not a transplant candidate due to multiple organ failure. As per family request, patient was palliatively extubated and died. Terbinafine is a fungicidal drug with activity against dermatophytes including Epidermophyton flccosum and trichophyton rubrum. It works by inhibition of squalene epoxidase with a resultant accumulation of squalene in the fungal cell and killing it as a result. Commonly used orally to treat onychomycosis and other fingernails and toenails infections. Shortly after its introduction to the market, DILI had been reported with elevation with serum aminotransferases elevation that was usually self-limited. Usually presents within first 6 weeks of therapy with either hepatocellular or cholestatic initially with sings of hypersensitivity. Mechanism of injury entails hypersensitivity reaction, though the full pathogenesis was not elucidated yet, but genetic polymorphism is implicated in the variable presentation especially among HLA-A 33:01 allele carriers. Terbinafine DILI resolves usually within 6 months of stopping the medication but can lead to death or need liver transplantation in some cases.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24530号 / 医博第4972号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小濱 和貴, 教授 小林 恭, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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)

An in vitro system has been developed to study N-acetyltransferase (NAT) activity using human liver slices in dynamic organ culture. Acetylation of para-aminobenzoic acid (PABA) and sulfamethazine (SMZ) in the presence of human liver slices was monitored by measuring the disappearance of the parent amine from the incubation medium using the colorimetric procedure of Bratton & Marshall. Presence of the acetyl conjugate was confirmed using HPLC. PABA acetylation rates varied from 0.72-2.52 nmoles/hr/mg protein (n = 8). This small variation (4 fold) is consistent with the classification of PABA as a monomorphic substrate. The variation in the rate of SMZ acetylation was greater than 20 fold (0.144-3.68 nmoles/hr/mg protein; n = 9). This larger variation is characteristic of SMZ as a polymorphic substrate. The results obtained indicate that human liver slices in dynamic organ culture can be used for the determination of hepatic NAT activity and acetylator status of individual human livers.

Long thought to be irreversible, it is now clear that liver fibrogenesis is a dynamic process, with scar tissue capable of being remodelled as well as deposited. Macrophages have been shown to have a critical role in both liver fibrogenesis and fibrosis resolution. Whilst previous work has identified a Ly-6Chi hepatic macrophage population, derived from recruitment of inflammatory monocytes, as being the main pro-fibrogenic population, the nature and phenotype of the pro-resolution macrophage subset is unknown. In this thesis, I sought to identify and characterise this restorative hepatic macrophage. I established a reversible murine model of liver fibrosis using CCl4. At the time of initiation of fibrosis regression, Ly-6Clo CD11bhi F4/80int hepatic macrophages represented the most numerous macrophage population and the principal expresser of matrix degrading MMP enzymes. Depletion of this population in CD11b-diphtheria toxin (DTR) mice prevented fibrosis resolution. Subsequent, adoptive transfer and in situ labelling experiments, demonstrated that this restorative macrophage population derives from inflammatory monocytes, a common origin to the pro-fibrotic Ly-6Chi hepatic macrophage subset, indicating a switch in macrophage phenotype in situ to form the restorative phenotype. Characterisation of FACS-sorted restorative and pro-fibrogenic liver macrophage subsets using gene expression profiling demonstrated higher expression of pro-resolution genes and lower expression of pro-fibrotic genes in restorative macrophages, which also upregulated a number of genes involved in phagocytosis. Confocal microscopy confirmed that restorative macrophages showed evidence of prior phagocytosis. This could be replicated in vitro, where feeding macrophages with cellular debris resulted in matrix-degrading properties analogous to those seen in vivo, which was dependent on activation of the ERK signalling cascade. This effect was also demonstrated with the phagocytosis of liposomes in vitro. Finally, the administration of liposomes to CCl4-injured mice in vivo induced phagocytosis, causing an increase in hepatic restorative macrophage number and accelerating fibrosis regression. Hence, I have been able to identify and characterise the restorative hepatic macrophage and have utilised these data to develop a novel method to alter macrophage phenotype in vivo and accelerate the resolution of liver fibrosis and restoration of normal tissue architecture.

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences