published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Acute Liver Failure (ALF) is a devastating ailment with a high mortality rate and limited treatment alternatives. This study presents a methodology for the design and development of a bio-artificial bioreactor to be used in a Bio-Artificial Liver Support System. The system will ultimately be used either to bridge a patient to orthotopic liver transplant (OLT), the only current cure for end stage ALF, or spontaneous recovery. Methods to optimize and visualize the flow and related mass transfer in the BR are presented. The use of magnetic resonance imaging (MRI), scanning electron microscopy (SEM) and simple testing methodology is applied with emphasis on modeling the flow conditions in the BR. The bioreactor (BR) used in the Bio-Artificial Liver Support System (BALSS), currently under-going animal trials at the University of Pretoria, was modeled and simulated for the flow conditions in the device. Two different perfusion steps were modeled including the seeding of hepatocyte cells and later the clinical perfusion step. It was found that the BR geometry was not optimal with “dead spots” and regions of retarded flow. This would restrict the effective transport of nutrients and oxygen to the cells. The different perfusion rates for the seeding and clinical perfusion steps allowed for different velocity contours with cells seeing inconsistent flow patterns and mass transfer gradients. An optimized BR design is suggested and simulated, that effectively reduces the areas of retarded flow (dead spots) and increases the flow speed uniformly through the BR to an order of magnitude similar to that found in the sinusoidal range. The scaffolding volume was also decreased to allow a larger local cell density promoting cell-cell interaction. Finally a summarized design table for the design of a hepatic BR is presented. / Dissertation (MEng (Mechanical))--University of Pretoria, 2008. / Mechanical and Aeronautical Engineering / unrestricted

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In this thesis I examined molecular mechanisms involved in acute and chronic liver injury, and also studied basic pathways mediating tumour promotion. Acute hepatic failure secondary to paracetamol poisoning is associated with high mortality. C-jun (NH2) terminal kinase (JNK) is a member of the mitogen activated protein kinase family and is a key intracellular signaling molecule involved in the control of cell fate. Paracetamol induced hepatic JNK activation in both human and murine paracetamol hepatotoxicity, and in a murine model preceded the onset of hepatocyte death. JNK inhibition in vivo (using two JNK inhibitors with different mechanisms of action) markedly reduced mortality in murine paracetamol hepatotoxicity. In addition, delayed administration of JNK inhibitor was more effective than N-acetylcysteine following paracetamol poisoning in mice. JNK inhibition was not protective in acute carbon tetrachloride or anti-Fas antibody mediated hepatic injury, suggesting specificity for the role of JNK in paracetamol hepatotoxicity. Furthermore, disruption of the JNK1 or JNK2 genes did not protect against paracetamol-induced hepatic damage. Pharmacological JNK inhibition had no effect on paracetamol metabolism, but markedly inhibited hepatic TNF-alpha production following paracetamol poisoning. These data demonstrate a central role for JNK in the pathogenesis of paracetamol induced liver failure, thereby identifying JNK as an important therapeutic target in the treatment of paracetamol hepatotoxicity. Liver fibrosis with loss of tissue architecture and subsequent hepatic failure represents a massive healthcare burden worldwide. Expression of Galectin-3 (a beta-galactoside binding animal lectin) is upregulated in established human fibrotic liver disease, during the development of experimental liver fibrosis and is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis. Disruption of the gene encoding Galectin-3 blocks transdifferentiation of precursors to myofibroblasts in vitro and in vivo, markedly attenuating hepatic scarring in a murine model of liver fibrosis. Inhibition of Galectin-3 expression by siRNA in primary murine and human hepatic stellate cells significantly reduced myofibroblast activation and procollagen(I) expression. The reduction in hepatic fibrosis observed in the Galectin-3-/- mouse occurred despite equivalent liver injury and inflammation, and similar tissue expression of TGF-beta. TGF-beta failed to transactivate Galectin-3-/- hepatic stellate cells, in contrast with wild type hepatic stellate cells. However TGF-beta stimulated signaling via Smad-2 and 3 was equivalent in both Galectin-3-/- and wild type hepatic stellate cells indicating that Galectin-3 is required for TGF-beta mediated myofibroblast activation and matrix production. This supports a novel and important mechanistic role for Galectin-3 in the regulation of myofibroblast activation and consequent liver fibrosis. Finally, in vivo siRNA knockdown of Galectin-3 inhibited myofibroblast activation following hepatic injury and may therefore provide a novel therapeutic approach to the prevention and treatment of liver fibrosis. CD98hc (a ligand for Galectin-3) constitutively and specifically associates with beta1 integrins and is highly expressed on the surface of human tumour cells irrespective of the tissue of origin. CD98hc promotes both anchorage- and serum-independent growth. Using chimeras of CD98hc and the type II membrane protein CD69 demonstrated that the transmembrane domain of CD98hc is necessary and sufficient for integrin association in cells. Furthermore, CD98hc/β1 integrin association is required for focal adhesion kinase-dependent phosphoinositol 3-hydroxykinase activation and cellular transformation. Amino acids 82-87 in the putative cytoplasmic/transmembrane region appear to be critical for the oncogenic potential of CD98hc and provide a novel mechanism for tumour promotion by integrins.

The Best MPhil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2003-2005 / published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

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.

Concerning potential immunological reaction in patients receiving multiple bioartificial liver treatments (with porcine hepatocytes bioreactor), the beneficial effect of sequential plasmapheresis-hemoadsorption-bio-liver treatment protocol was studied. The hypothesis was tested in a trichosanthin-induced anaphylaxis rat model. Fatal anaphylactic response upon antigen challenge was reduced in a previously trichosanthin sensitized rat prior with cross circulation. It was suggested that circulating antibodies responsible for immunological response were removed by the non cell-based treatment. The potential risk of hypersensitivity reaction of subsequent cell-based treatment was reduced. In the design of HBLSS, the sequential plasmapheresis-hemofiltration-bio-liver treatment was suggested to alleviate potential immunological consequences and allow multiple uses in single patient. / In addition to artificial liver research, pharmacological therapies are sought to treat ALF. Recent studies suggested that the local renin-angiotensin system (RAS) in the liver regulate the fibrogenic response. The possible involvement of RAS in acute liver injury was investigated in the present study. In the D-galactosamine induced rat liver failure model, angiotensin II type 1 receptors (AT1R) were detected by immunohistochemical staining in the centrilobular region after induction of acute liver injury which was not evident in normal liver. There were associated elevation of total bilirubin, alanine aminotransferase and tissue inhibitor of metalloproteinase type 1 (TIMP-1). Losartan treatment was able to reduce all these parameters. TIMP-1 protein was reduced by 1.5 fold (p<0.05) on day 1 and 1.56 fold (p<0.05) on day 3 in the losartan treatment group relative to the GalN group. The survival rate of the losartan treatment group was significantly higher than that without treatment (5-day survival, 85% vs 42.5%, p<0.05). This finding suggested the local renin-angiotensin system plays a role in the pathophysiological mechanism of acute liver injury. / In summary, the presence study addressed two approaches in treating liver failure. The HBLSS treatment protocol improved survival of acute-on-chronic liver failure patients. The protective effect of losartan in liver injury suggests AT1R blockade is a potential therapeutic strategy in acute liver injury. / The mainstay treatment of acute liver failure (ALF) is conservative medical therapy. In patients having acute liver insufficiency, the only proven life-saving procedure is liver transplantation. The concept of supportive care in liver failure is to optimize patient's clinical condition by replacing some of the essential functions of liver and allowing liver regeneration in order to compensate the loss of hepatocellular functions. / There was increasing evidence suggesting a role of artificial liver (extracorporeal system) in the treatment of liver failure. A retrospective study was performed in a group of acute-on-chronic liver failure (AoCLF) patients treated with hybrid bioartificial liver support system (HBLSS). From 2001-2004, there were 40 chronic liver disease patients presented with acute deterioration. Patients were treated either with conventional medical therapy (n=21) or integrated with HBLSS treatment (n=19). The 60-day survival rate was 30% and 68.5% respectively (P<0.05 log-rank test). Integration of hybrid bioartificial liver support system (HBLSS) in treating acute-on-chronic liver failure (AoCLF) patients improved the survival outcome. Univariate analysis of admission blood parameters revealed creatinine appeared to predict mortality in AoCLF patients with HBLSS treatment. / Chan Hoi Ming Herman. / "June 2006." / Adviser: Siu-cheung Michael Tam. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1547. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 104-116). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Infections in patients with acute liver failure (ALF) and acute liver injury (ALI) are a frequent occurrence. Because ALF and ALI patients share many of the same clinical features as patients with severe sepsis and septic shock, identifying an infection based upon clinical manifestations is extremely difficult. Bacterial culture and sensitivity reports require 24 to 72 hours to be finalized after the need for a culture is suspected and obtained. During this time period, ALF and ALI patients are either not receiving required antibiotic therapy, receiving antibiotic therapy that is not required or not appropriate for the infecting bacterial pathogen, or receiving the correct antibiotic prophylaxis. Receiving an antibiotic that is not needed or inappropriate adds another level of complexity to the ALF and ALI patients because antibiotics may exacerbate liver dysfunction. The purpose of this study was to determine the utility of serum procalcitonin concentrations (SPCTC) as a biomarker of bacterial infections in patients with acute liver failure (ALF) and acute liver injury (ALI). This three part study measured SPCTC retrospectively on samples from ALF and ALI patients who were prospectively enrolled in the United States Acute Liver Failure Study Group (USALFSG) ALF and ALI studies. In the first part of the study, subjects were categorized according to how many SIRS continuum components they had and whether there was a documented infection. In the second part, serial samples on subjects who developed infections were identified. And, in the third part, serial samples on subjects diagnosed with infection on day one of the study and categorized based upon transplant free survival (TFS) or death and/or liver transplant (DoT) were identified. Procalcitonin was not found to be useful in identifying infection in the ALF and ALI patient populations. A cut-off for indication of infection was calculated to be 1.62 ng/mL using receiver operator curve (ROC) analysis. Despite the fact that there was an overall increase in SPCTC as the severity of illness increased in patients with a documented infection, there were confounding variables including antibiotic use, missing data, and small sample size that may have contributed to the poor sensitivity and specificity (0.643 and 0.620 respectively) calculated as part of the ROC analysis. SPCTC values appeared to be increased in subject with acetaminophen (APAP) toxicity and may have affected the cut-off, sensitivity, and specificity results. Increased SPCTC values were seen in APAP subjects who did not have a documented infection. It is unknown at this time if the SPCTC were increase due to liver damage, an undiagnosed infection, or as a result of increase cytokine production due to the APAP toxicity. Serial PCT concentrations in patients who achieved TFS showed a greater decrease over time than those of patients who died or received a liver transplant, however, the TFS group contained a large portion of APAP subjects. Further prospective studies are needed to determine the extent of interference with SPCTC in patients with APAP toxicity and to better define the PCT concentration cut-off between infection and no infection in the ALF and ALI populations.