Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

published_or_final_version / Pathology / Master / Master of Medical Sciences

Background: Hepatocellular carcinoma is one of the most fatal diseases worldwide. Liver transplantation dramatically improved the survival rate of HCC patients. However, tumor recurrence remains a huge threat to HCC patients without any promising curative treatment. Chemotherapy, as one of the potential treatments to recurrent HCC, did not show any significant effect either. Objective: We aim to investigate the role of interferon-gamma inducible protein 10 (IP10) in acute-phase liver graft injury induced late-phase cisplatin resistance after liver transplantation and to explore the underlying mechanism. Furthermore, a potential adjuvant therapy was expected to be identified to sensitize cisplatin treatment in HCC. Materials and methods: A rat orthotopic liver transplantation model was established with applying whole or small-for-size (50%) graft. Afterwards, a rat hepatoma cell (MH7777) was injected via portal vein to generate recurrent tumor. The expressions of genes linked to multi-drug resistance and graft injury were compared between tumors developed after liver transplantation using small and whole grafts. IP10 expression was further validated in clinical samples from two cohorts of patients including HCC patients with hepatectomy and HCC patients with liver transplantation. The extracellular and intracellular roles of IP10 were examined in vitro by using IP10 recombinant protein and IP10 stable transfectants in HCC cell lines. The correlation between IP10 expression and tumor growth was investigated in three in vivo nude mice models including a subcutaneous model, an orthotopic model and ischemia reperfusion injury model. The underlying mechanism was further explored in vitro, in vivo and in clinical samples. IP10 neutralizing antibody was employed as an adjuvant therapy to identify its effect on sensitizing cisplatin treatment in HCC. Results: The expressions of multidrug resistant genes were significantly up-regulated in liver and tumor from small-for-size group in rat liver transplantation model. IP10 was selected as the potential target for its constantly higher expression in liver and tumor tissues in small-for-size group. In clinical studies, IP10 was overexpressed in around 45% HCC patients with hepatectomy. The expression of circulating IP10 well correlated with tumor recurrence and small graft ratio in HCC patients after liver transplantation. In in vitro studies, it was demonstrated that overexpression of IP10 could significantly promote HCC cell proliferation either in short term or in long term cisplatin administration. In in vivo studies, subcutaneous and orthotopic nude mice models showed that the overexpression of IP10 have significant correlations with larger tumor volume and less tumor necrosis after cisplatin treatment. In mechanism studies, IP10 overexpression was found to be well correlated with the activation of endoplasmic reticulum (ER) stress signaling pathways in vitro and further validated in vivo models and in clinical specimens. IP10 neutralizing antibody was identified as a potential therapy which could sensitize cisplatin treatment in vitro and in vivo. Conclusions: The high expression of IP10 was identified in two cohorts of clinical samples and showed significant correlations with tumor recurrence. Graft injury induced IP10 overexpression could significantly increase cisplatin resistance after liver transplantation via ER stress signaling pathways. IP10 neutralizing antibody may be applied as an alternative treatment for recurrent HCC after liver transplantation. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Recruitment of effector T cells to sites of infection is essential for an effective adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis describes the signaling events invoked by CCL5 and its ability to regulate the energy status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in a dose-dependent manner. Collectively, these studies suggest that CCL5 may also influence the metabolic status of activated T cells by simultaneously activating the AMPK and mTOR pathways.

Severe acute respiratory syndrome (SARS) was defined as an invasive respiratory disease in 2002, which originally came from China and rapidly spread all over the globe. Acute pneumonia and lower respiratory tract involvement most affected the middle aged individuals and elderly with a mortality rate of 11%. While SARS Corona virus (SARS-CoV) has maintained its potential capacity to reemerge, clinical study of the immune system of SARS patients, as well as controlled studies may lead to application of new treatment strategies in future. Throughout this work, I have focused on early immune responses to SARS-CoV in humans and in ferrets. CXCL0 has been associated with alterations in the clinical course of several infectious diseases, including SARS and influenza. Here I have cloned ferret CXCL10 gene and have expressed its recombinant protein. I demonstrate that the CXCL10 plasma level in SARS patients is associated with the severity of disease. I also show that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the lungs of deceased SARS patients and ferrets experimentally infected with SARS-CoV. Type I interferons (IFNs) are indispensable parts of the innate immunity during early stages of infection. A clear distinction between genes upregulated by direct virus-cell interactions and genes upregulated by secondary IFN production has not been made yet. Here, I have investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-a2b and during SARS-CoV infection. In vivo experiments revealed that IFN-a2b causes upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte migration. SARS-CoV infection of ferrets leads to upregulation of varieties of IRGs and a broad range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV infection, which are part of a robust IFN antiviral response. Since localization of CD8+ Tcells may contribute to tissue injury, I have characterized ferret CD8 gene and have generated reagents that can be used in future studies with the aim of evaluating CD8+ T cells localization in the ferret lung during infection with SARS-CoV.

Recruitment of effector T cells to sites of infection is essential for an effective adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis describes the signaling events invoked by CCL5 and its ability to regulate the energy status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in a dose-dependent manner. Collectively, these studies suggest that CCL5 may also influence the metabolic status of activated T cells by simultaneously activating the AMPK and mTOR pathways.

Respiratory infection remains a leading cause of morbidity and death in severely immunocompromised febrile neutropenic haematology patients, despite the introduction of numerous prophylactic strategies and advances in diagnosis and treatment. Prognosis is improved if an organism can be isolated and specific therapy commenced as soon as possible. Current practice in this population group is to commence empirical antibiotics and perform flexible bronchoscopy (FB) if temperature does not settle or after patients develop clinical or radiological features suggesting a respiratory source. This delay may result in a lower procedural diagnostic yield due to prior or prolonged anti-microbial treatment, and increased risk of respiratory compromise and procedural complications due to advanced respiratory infections. We hypothesised that proceeding to FB as early as possible after developing febrile neutropenia would improve treatment outcomes. With this randomised, prospective trial, we aim to further define the role of FB with reference to optimal timing of the procedure and its impact on diagnostic yield, future management and complication rate. We also aim to analyse the impact of proven infection on the cytokine profile of immunocompromised patients. Methods: Patients with acute leukaemia, allogeneic bone marrow transplantation or chronic lymphocytic leukaemia (CLL) being treated with Fludarabine/ Mabthera without an obvious non-respiratory source of infection were prospectively randomised into early bronchoscopy or conventional management groups at onset of febrile neutropenia. Bronchoalveolar lavage (BAL) fluid chemokine levels (IP-10, RANTES, MIG, IL-8, MCP-1) were measured using a human Chemokine cytometric bead array (CBA) kit. Results: Thirty-one episodes of febrile neutropenia in 29 patients were analysed; 17 conventional and 14 early. There was an increased yield in fungal growth in the early bronchoscopy group, which was not predicted by prior clinical or radiological changes. However, this had no impact on clinical management in the short-term due to the delayed growth. Overall diagnostic yield was not significantly different between the two groups. Procedural complication rate was negligible overall and there was no difference associated with either group. IP-10 and MIG were significantly lower in those patients who had a fungal pathogen isolated, compared with those study patients who did not (175.17 vs 1157.8, p=0.03, 30.33 vs 247.8, p=0.03 respectively). IP-10 levels were higher in the conventional than early group (1253.0 vs 261.14, p = 0.035) and the study population had higher MCP-1 (734 vs 2.83, p=0.006) and IL-8 levels (606.9 vs 14.25, p=0.00655) than normal controls. Those cases with fungal infection had higher mean MCP-1, RANTES and IL-8 levels than in normal controls (844.0 vs 2.83, p=0.007; 17.5 vs 2.1, p=0.03; 156.0 vs 14.25, p=0.004). Conclusions: Early bronchoscopy as a component of the septic screen in febrile neutropenic patients was feasible and safe. A significant difference in fungal yield was seen in the early bronchoscopy group compared to conventional methods, with a negligible complication rate, but this did not result in a change in immediate clinical management or outcomes. / Thesis (M.Clin.Sc.) - University of Adelaide, School of Medicine, 2009

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Institute of Immunology and Allergy Research, Westmead Millennium Institute, Westmead Hospital, University of Sydney, 2003. / Bibliography: leaves 177-222.

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001? / Copy of author's previously published work inserted. Bibliography: leaves 139-160.