Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 164-173). Also available online.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 164-173). Also available in print.

Small cell lung cancer (SCLC) is characterized by prompt response to chemotherapy and radiotherapy but relapsing with drug resistance and distant metastasis, leading to poor overall prognosis. New anticancer agents and regimens are drastically needed for SCLC treatment. Arsenic trioxide (ATO), a traditional Chinese medicine used as a poison for thousands of years, has been tested in many hematological and solid cancers both in vitro and in vivo, with promising effects. In order to establish the scientific ground for future clinical application of ATO in SCLC, this study aimed to investigate the anticancer effect and mechanism of ATO in SCLC using in vitro and in vivo models, either as a single agent or in combination with standard chemotherapy. In addition, an ATO-acquired resistant cell line (H841-AR) derived from SCLC cell line H841 was used to explore potential mechanisms of ATO resistance and cross-resistance to other chemotherapeutic drugs. In the first part of this study, ATO was shown to exert cytotoxic effect in all of the chosen SCLC cell lines. Various cellular mechanisms were triggered upon ATO exposure: redox status disturbances (hydrogen peroxide (H2O2) generation, glutathione (GSH) depletion and thioredoxin 1 (Trx1) down-regulation), mitochondrial membrane depolarization (MMD), DNA damage, apoptosis and necroptosis. In concert with this, Bcl-2 was down-regulated accompanied by MMD, release of AIF and SMAC, DNA degradation, XIAP inhibition and caspases activation. Adoption of N-acetyl-L-cysteine (NAC) and buthionine sulfoximine (BSO) demonstrated GSH depletion and reactive oxygen species (ROS) generation played the pivotal role to mediate cytotoxic effect of ATO in SCLC. In the second part of this study, when combined with chemotherapeutic agents, ATO displayed synergistic and antagonistic interaction with cisplatin and etoposide respectively in SCLC cell line models. The beneficial combination of ATO and cisplatin was also substantiated by tumor xenograft models. Augmented GSH depletion and suppressed drug efflux mechanism were found to explain the synergistic effects. In the last part of this study, H841-AR was generated as an acquired multi-drug resistant (ATO, cisplatin and etoposide) cell line to investigate the potential resistance mechanisms and possible future drug combinations. Comparing H841-AR cells with parental H841 cells using cDNA microarray, a long list of genes was altered in ATO-resistant cells. At least 20 up-regulated and 45 down-regulated genes were short-listed as candidates with a cut-off at 5-fold change. Interestingly, qPCR data has shown that 5 selected up-regulated genes in H841-AR cells were also highly expressed in DMS79 cells with intrinsic ATO resistance compared to the relatively sensitive cell lines, indicating that these genes might be associated with ATO resistance in SCLC. In summary, ATO was shown to be an active anticancer agent in SCLC, either alone or in combination with cisplatin. The major mechanisms of action of ATO and its synergism with cisplatin in SCLC were elucidated. Genetic data derived from an acquired resistant (to ATO, cisplatin and etoposide) SCLC cell line may help to uncover the mechanisms of resistance to ATO, allowing possible future drug combinations. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

There is no rapid diagnostic technique at medical examiners' offices to determine if a decedent is infected with Mycobacterium tuberculosis. Present diagnostic testing requires at least one month for results. A biosensor-based sandwich immunoassay for the detection of M. tuberculosis was developed in this study. M. tuberculosis polyclonal antibody was used for target antigen capture and detection in the immunoassay. Live attenuated M. tuberculosis (ATCC 25177) suspended in phosphate-buffered saline with 0.1% Tween 20 was used as the antigen in the detection assay. The Analyte 2000 was the initial biosensor platform. Initial testing was of Freund's adjuvant complete. M. tuberculosis was detected 50% of the time at 1,000,000 CFU/ml and 100% of the time at 10,000,000 CFU/ml and 100,000,000 CFU/ml. Live attenuated M. tuberculosis was also tested using the Analyte 2000 biosensor. Detection was obtained 87.5% of the time at 1,000,000 CFU/ml and 100% of the time at 10,00 0,000 CFU/ml and 100,000,000 CFU/ml. The RAPTOR, an automated, portable instrument, was then tested as the fiber optic biosensor platform. Positive biosensor detection was obtained 75% of the time at cell concentrations of 1,000,000 CFU/ml, 95% of the time at 10,000,000 CFU/ml, and 99% of the time at 100,000,000 CFU/ml. Live attenuated M. tuberculosis suspended in PBST and seeded into decedent lung tissue was tested using the RAPTOR. Positive detection was obtained 21% of the time at cell concentrations of 1,000,000 CFU/ml, 86% of the time at 10,000,000 CFU/ml and 100% of the time at 100,000,000 CFU/ml. Antibody specificity studies using ELISA were performed to determine the anti-M. tuberculosis antibody's cross reactivity with microorganisms other than M. tuberculosis. M. tuberculosis actively growing in the lung of an individual is found at levels of 10,000,000 to 1,000,000,000 CFU in the lesions of the lung. This study determined that the RAPTOR biosensor assay was capable of detecting the presence of M. tuberculosis in lung tissue homogenate within three hours when the concentration of M. tuberculosis was 10,000,000 to 1,000,000,000 CFU/ml.

Kinder, welche nach 34 (+0) bis 36 (+6) Gestationswochen geboren werden, bezeichnet man als späte Frühgeborene. Genau wie Kinder eines jüngeren Gestationsalters sind diese von einer höheren postnatalen Morbidität und Mortalität betroffen als reif geborene Kinder. Diese Studie betrachtet die pulmonale Funktionsleistung dieser Kinder weit über die Neonatalperiode hinaus. Ehemals späte Frühgeborene wurden im Alter von 6 Jahren untersucht. Eine gleichaltrige Kontrollgruppe, bestehend aus ehemals reifen Neugeborenen, diente dem Vergleich. Vor allem Parameter der Spirometrie sowie Peak-Flow- und Atemwegswiderstandsmessungen ermöglichten dabei die Objektivierung der pulmonalen Funktion. Statistisch signifikante Unterschiede ließen sich in der mittleren FVC (forcierte Vitalkapazität) sowie dem FEV1 (forciertes exspiratorisches Volumen in einer Sekunde) feststellen, wobei Kinder der Indexgruppe jeweils im Mittel nur geringere Werte erreichten als Kinder der Kontrollgruppe. Der mittlere Atemwegswiderstand unterschied sich in Index-und Kontrollgruppe ebenso signifikant, wobei bei ehemals späten Frühgeborenen der Atemwegswiderstand im Mittel höher war als bei ehemals reifen Neugeborenen.

Lung cancer is the leading cause of cancer deaths worldwide. A large part of this high mortality rate is due to the onset of metastatic disease prior to diagnosis. Advances in treatment for metastatic disease may be achieved by understanding more about the identity of metastatic tumor cells and the mechanisms those cells employ to spread throughout the body. This thesis examined the relationship between cells capable of tumor propagation upon serial transplantation (tumor-propagating cells, or TPCs) and those with metastatic potential.

Macrophages are cells of the innate immune system that can be found in large quantities in cancer tumors and affect cancer progression by regulating growth and invasiveness of cancer cells. There are two main phenotypes of macrophages denoted M1 and M2. In this thesis, the M1 and M2 phenotype of human macrophages were characterized, and effects of the macrophages on the growth and invasiveness of colon and lung cancer cells were studied. Macrophages of the M1 phenotype, but not the M2 phenotype, inhibited growth of both colon and lung cancer cells, and the inhibition for some of the cancer cell lines was induced by cell cycle arrest in the G1/G0 and/or G2/M cell cycle phases. In the colon cancer cell line, the macrophage induced cell cycle arrest was found to attenuate the cytotoxic effect of the chemotherapeutic drug 5-FU. Macrophages were also shown to express high levels of proteases (matrix metalloproteinase-2 and 9) and high levels of proteins of the urokinase-type plasminogen activator (uPA) system, in comparison to the lung cancer cell lines studied. Expression of these has been found to predict poor outcome in lung cancer, and the results suggest macrophages to be important contributors of these in lung tumors. Furthermore, the M1 phenotype was found to express higher levels of the uPA receptor than the M2 phenotype. Prostaglandin E2 (PGE2) is a potent inflammatory molecule expressed by e.g. macrophages and monocytes, and inhibition of its expression has been shown to reduce the risk of colon cancer. Green tea and black tea was found to be potent inhibitors of PGE2 formation in human monocytes, and the inhibitory effects of green tea was likely due to its content of the polyphenol epigallocatechin gallate. Rooibos tea also inhibited PGE2 formation, but was less potent than green and black tea. The primary mechanism for the inhibition was via inhibition of expression of enzymes in the PGE2 formation pathway, and primarily microsomal prostaglandin synthase-1. / Macrophages are cells of the immune system often found in large numbers in cancer tumors. They affect multiple aspects of cancer progression, including growth and spread of cancer cells, and the efficacy of treatments. There are two major macrophage phenotypes denoted M1 and M2, that have mainly pro- and anti-inflammatory properties, respectively. In this thesis, M1 and M2 macrophages were characterized and effects of them on different aspects of cancer progression were studied using culture of colon, and lung cancer cells. The M1 phenotype inhibited proliferation of cancer cells from both colon and lung. The growth inhibition was for some cell lines accompanied by cell cycle arrest. The macrophage induced cell cycle arrest was found to protect colon cancer cells from the cytostatic drug 5-fluorouracil. Prostaglandin E2 (PGE2) contributes to colon cancer development and treatment of monocytes with tea extracts inhibited PGE2 formation via inhibition of expression of microsomal prostaglandin E synthase-1. Proteases can degrade the extracellular matrix of a tumor to facilitate cancer cell invasion and metastasis. The M1 and M2 phenotypes of macrophages expressed several protease activity related genes to a greater extent than lung cancer cells, and M1 more so than the M2 phenotype.

Since the discovery of cyclosporine in 1971, calcineurin inhibitors have played a critical role in the therapeutic suppression of the immune response. Patients receiving solid organ transplants rely heavily on these medications to prevent the acute and chronic rejection of allografted tissue. Introduction of tacrolimus, the most frequently prescribed calcineurin inhibitor, has lead to improved clinical outcomes for organ transplant recipients; however, little improvement has been noted in lung transplantation. Difficulties with current oral dosing regimens for lung transplant patients stem primarily from drug systemic toxicity, heightened risk of invasive infection, and erratic oral bioavailability. We have proposed that pulmonary delivery of a tacrolimus formulation with improved solubility can provide high lung concentrations, while limiting corresponding systemic levels associated with toxicity. Chapter 2 investigates the pulmonary administration of tacrolimus dispersion for nebulization to lung transplanted rats. Resulting lung and blood levels were determined by appropriate bioanalytical methods. Limited systemic absorption was seen after pulmonary delivery, resulting in a 50 to 1 lung to blood concentration ratio. A 28 day safety and stability evaluation of tacrolimus dispersion for nebulization was conducted in Chapter 3. Results showed no signs of toxicity in Sprague Dawley rats and proved the stability of tacrolimus powder for dispersion for 3 months. For cases of severe asthma, immunosuppression is also necessary to restore normal lungs function and is typically treated with corticosteroids. Corticosteroids, however, are well known for their untoward side effects and can prove ineffective in severe asthmatics that have developed corticosteroid resistance. Chapter 4 investigates the use of tacrolimus dispersion for nebulization for prophylactic treatment of asthma. Efficacy was determined in an asthma-induced animal model by quantification of inflammatory cells and signaling chemicals. In Chapter 5, tacrolimus powder for inhalation is investigated in a novel dry powder inhalation platform. Respirable particles are produced when bulk particles (500 [micrometer]) comprising a matrix of drug/excipient are sheared apart by a marketed inhalation device to produce particles of the appropriate geometric diameter (50 [micrometer]). Biocompatible material with brittle properties were found to produce fine particle fractions (FPF) up to 70.3% and total emitted doses (TED) higher than 95%. / text