Multidrug resistance protein-1 (mdr1) is a well characterized membrane protein, expressed in a variety of cell types. In cancer cells, an overexpression of mdr1 has the function of conferring drug-resistance. The exact physiological function of mdr1 constitutively expressed in normal cells still remains unclear. A goal of this work was to determine if there is an increase in expression of mdr1, above constitutive levels, on CD4+ and CD8+ T-lymphocytes, following cardiac transplantation. The expression of mdr1 was correlated with episodes of acute cardiac rejection in order to determine if mdr1 has the functional ability to confer immunosuppressive drug resistance. Drug resistant CD4+ and CD8+ T-lymphocytes could explain episodes of acute allograft rejection observed in a number of immunosuppressed patients. Immunochemical techniques measuring mdr1 were performed on endomyocardial biopsy specimens and peripheral blood mononuclear cells (PBMCs) isolated from cardiac transplant patients. Functional mdr1 assays and flow cytometry were performed on PBMCs isolated from cardiac transplant patients prior to transplantation and at longitudinal time points post-transplantation. The expression and function of mdr1 was correlated to the histological diagnosis of acute rejection. Immunochemical analysis of endomyocardial biopsy samples showed mdr1 expression localized on the plasma membrane of graft infiltrating mononuclear cells. Immunochemical analysis of PBMC samples showed a constitutive mdr1 expression on normal volunteer PBMCs and a increased expression of mdr1 on cardiac transplant PBMCs during episodes of acute rejection. Doxorubicin cytotoxicity assays showed an increased drug resistance profile in transplant patients compared to normal individuals. An mdr1 efflux assay demonstrated an increase efflux function in PBMCs during episodes of acute rejection. Flow cytometric analysis showed significant increases in the intensity of mdr1 expression on CD4+ and CD8+ T-lymphocytes in transplant patients compared to normal individuals. Flow cytometry also confirmed a significant increase in both the number of CD4+ and CD8+ T-lymphocytes expressing mdr1 and the intensity of mdr1 on these subtypes for those patients that had an episode of acute rejection compared to those transplant patients with no episodes of acute rejection. Thus, this study demonstrates an overexpression of a functionally active multidrug resistant protein-1 during episodes of acute rejection.

Increasing evidence implicates the role of reactive oxygen species in the pathogenesis of several diseases such as cardiovascular disease. Recent studies also suggest a role for reactive oxygen species in the control of vascular smooth muscle proliferation both in vitro and in vivo. By altering the balance in the endothelium between vasoconstrictors such as thromboxane, isoprostanes and vasodilators such as nitric oxide, reactive oxygen species contribute to endothelium-dependent contractions and increased vascular resistance. Studies in animal models of atherosclerosis suggest that natural and synthetic antioxidants can retard the development of atheroma. Antioxidants can restore endothelial function and decrease blood pressure in several models of hypertension and in some essential hypertension of humans. Epidemiological comparisons between populations and studies within populations also support the contention that high plasma levels or dietary intake of natural antioxidant may protect against the development of coronary disease in man. Endothelial dysfunction is increasingly recognized as an early event in the pathogenesis of cardiovascular disease. Endothelial dysfunction and coronary artery diseases are both linked to hypertension. Modification of these conditions improves both endothelial function and coronary artery disease outcomes. Dietary and lifestyle modifications and antioxidant vitamin supplementation have a beneficial effect on endothelial function, as do angiotensin-converting enzyme inhibitors and lipid-lowering agents. Oxidative stress increases cell proliferation, mediates hormone-induced hypertrophy, and under some circumstances-induces apoptosis. Smooth muscle cells contain a reduced nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide phosphate oxidase that is responsible for the majority of the superoxide produced by the vessel wall. This enzyme has been characterized biochemically, but only limited information is available regarding its molecular structure. High levels of oxidative stress are apparently involved in the pathogenesis of vascular diseases such as hypertension and atherosclerosis. Thus the pathways responsible for oxidative stress, as well as the antioxidant defenses in the vessel wall, may provide novel therapeutic targets.

Cell adhesion molecules (CAM) have been implicated in the process of cancer metastasis. However, the interactions of metastatic tumor cells with organ parenchymal cells-hepatocytes particularly the relevance of tumor-cell-hepatocyte adhesion to liver metastasis is still not fully understood. We have investigated the role of tumor cell-hepatocyte interaction in liver metastasis using liver metastatic subline H-59 of the Lewis lung carcinoma. A monoclonal antibody (MAb) C-11 was produced which was highly specific to liver metastatic H-59 cells. Our in vitro studies showed that this MAb and its F(ab)2 fragments could block tumor H-59 cell adhesion to hepatocytes. The molecule detected by MAb C-11 on H-59 cells as well as on hepatocytes was a 64-71 kDa plasma membrane glycoprotein (designated C-11 BP). We found that this molecule contains approximately 43% of N-linked carbohydrates which were not involved in MAb C-11 recognition, but participated in the adhesion process. MAb C-11 could also block the stimulatory effect of hepatocytes on tumor cell proliferation indicating that the adhesion of H-59 cells to hepatocytes may be necessary for tumor cell growth. The in vivo role of the C-11 BP was subsequently tested and it was shown that MAb C-11 and its F(ab)2 fragments could significantly reduce the number of hepatic metastases when used to pretreat H-59 cells before intrasplenic injection, or when inoculated directly into the animals by the intraperitoneal and intravenous routes. This inhibitory effect resulted in an increase in the survival time of tumor-inoculated mice. In additional studies, a second molecule namely the membrane-associated endoplasmic reticulum protein ERp-72 was also implicated in the adhesion of H-59 cells to the hepatocytes, however its role in vivo remains to be confirmed. Our results suggest that CAM involved in tumor-hepatocyte adhesion play an important role in liver metastasis. Monoclonal antibody against tumor cell surface adhesion molecul

Patients with mitochondrial myopathies characteristically exhibit pronounced exercise intolerance, often associated with lactic acidosis, tachycardia and muscle weakness. These clinical features are attributable to impaired electron transport chain function in skeletal muscle. The usual etiology is a primary defect in mitochondrial DNA (mtDNA), where the severity of impairment is presumably linked to the ratio of mutant to wild-type mtDNA. This dissertation presents novel therapeutic approaches to these genetic defects, aimed at attenuating mitochondrial dysfunction and ameliorating the clinical condition by employing exercise training alone or in conjunction with pharmacological therapy. Dichloroacetate (DCA) was administered to augment mitochondrial capacity by activating pyruvate dehydrogenase, thereby decreasing lactic acidosis. Endurance and resistance training paradigms were employed to induce mitochondrial and satellite cell proliferation respectively. The goals were to augment respiratory chain function, increase levels of wild type mtDNA, and reverse effects of chronic inactivity. The effects of these treatments on functional and mitochondrial capacity were defined by changes in: (1) work capacity, oxygen utilization, and circulatory responses during maximal exercise; (2) heart rate and blood lactate during submaximal exercise; (3) recovery kinetics of phosphate-containing metabolites measured using phosphorus magnetic resonance spectroscopy ( 31P MRS); (4) scores on a quality of life questionnaire. The cellular correlates for these indices were defined by changes in: (1) mitochondrial volume, (2) respiratory chain enzyme activity, and (3) levels of mutant/wild-type mtDNA. Although DCA administration alone lowered blood lactate, endurance training was more effective in improving exercise capacity, heart rate and blood lactate, 31P MRS recovery kinetics, and quality of life. Increased mitochondrial volume and respiratory chain function were closely linked

Introduction. Preliminary studies found that discordance between the patients' and physicians' assessments of disease activity in systemic lupus erythematosus (SLE) exists. We investigated the factors associated with discordance and explored the impact of discordance on medication adherence and complementary/alternative medicine (CAM) use. / Methods. Part I investigated the factors associated with discordance, defined as the patient visual analog scale (VAS) minus the physician VAS for global disease activity. Data were extracted from the Montreal General Hospital Lupus Registry. Potential covariates included the Medical Outcomes Studies SF-36, the Systemic Lupus Activity Measure (SLAM), and a lupus damage index. The first visit data were analyzed using multiple regression. Unbalanced repeated measures analysis of variance was used to analyze follow-up data and to investigate the influence of time. / Part II used a patient questionnaire to measure adherence and CAM use, which was then linked to discordance data from the Registry. The associations between discordance and non-adherence and CAM use were tested using multivariable logistic regression. Non-linear relationships were tested by generalized additive models (GAM). / Results. Clinically important discordance occurred in nearly 30% of the visits. The SF-36 scales for Bodily Pain and Vitality were important variables for predicting discordance. SLAM-Skin and -Musculoskeletal components were also associated with discordance. The mean discordance tended to increase over time. While both the patients' and physicians' VAS scores tended to decrease over time, the decrease was more pronounced in the physicians' VAS scores. / Non-adherence and CAM use occurred in 32% and 55% of the subjects, respectively. Patients who scored much lower disease activity than their physicians were more likely to be non-adherent than concordant patients (odds ratio = 2.25, 95% confidence interval: 0.32, 15.96). GAM testing supported this finding. Odds ratios for discordance and use of CAM therapies ranged from 0.89 to 1.48 (all non-significant), and GAM showed a non-linear relationship represented by an inverted U-shaped curve. / Conclusion. Patient-physician discordance exists in SLE. Factors such as bodily pain and fatigue increase discordance while clinically visible signs, such as skin manifestations, reduce discordance. Clinically important discordance appears to be associated with patient self-care behaviour, particularly, medication nonadherence.

<p> Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2^nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients). </p><p> The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. </p><p> Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease.</p>

Arterial thrombosis is one of the important pathophysiological mechanisms that lead to cardiovascular diseases. In this thesis, we have made an attempt to better characterize the kinetic and molecular mechanisms that underlie the critical first step in arterial thrombosis, namely, the interaction between platelet glycoprotein (GP) Ib and von Willebrand factor (VWF). In the first part of the work, we evaluated the kinetics of interaction between platelet GP Ib-IX-V complex and VWF under arterial flow conditions. The GP Ibalpha subunit of GP Ib complex binds to VWF through the Al domain of VWF. Impaired GP Ib-VWF interaction due to GP Ibalpha mutations can result in bleeding abnormalities including platelet-type von Willebrand disease (ptVWD). We measured the cellular on- and off-rate constants of CHO cells expressing wild-type or gain- or loss-of-function mutant GP Ibalpha interacting with VWF-Al-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on VWF-Al surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of VWF-Al, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency was 3-fold more and 3-fold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GP Ibalpha affect both the association and dissociation kinetics of the GP Ibalpha-VWF-Al bond. In the second part of the work, we compared the interaction of unusually large multimers of VWF (ULVWF) and that of the normal plasma multimers of VWF (P-VWF) platelets. ULVWF multimers are implicated in the pathology of a thrombotic disorder, thrombotic thrombocytopenic purpura (TTP) due to their increased affinity for platelets. We found that the ULVWF multimers are more effective than the normal P-VWF multimers in mediating (a) platelet aggregation in solution at high shear stress; (b) ristocetin-modulated platelet agglutination and (c) platelet adhesion to immobilized VWF under arterial shear conditions.

Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes meningitis in infants. Located in the outer membrane, Hib porin (342 amino acids, M$ rm sb{r}$ 37,782 daltons) forms channels that allow for the diffusion into the periplasm of small solutes up to a molecular mass of 1400 daltons. / Based on predictions of porin secondary structure, a model of Hib porin was generated. The model proposed sixteen membrane spanning $ beta$-strands connected by eight long loops on one side and eight short $ beta$-turns on the other side. To refine further this model, the epitopes of nine monoclonal antibodies specific for Hib porin were mapped precisely. Surface-exposure of these epitopes was determined by flow cytometry of intact Hib cells. Our studies identified two surface-exposed regions of Hib porin: amino acids 162 to 172, and 318 to 325. These two regions correspond to loops 4 and 8 in the Hib porin model. / Three naturally occurring Hib porin variants were purified, reconstituted into planar bilayers, and analyzed for their voltage dependency. Substitutions at Arg166 residue, which is localized to loop 4, were associated with a lowered threshold potential for the voltage gating of Hib porin. This surface-exposed loop was therefore implicated in the conformational changes that are postulated to occur during voltage gating. / Lipooligosaccharide (LOS) binds tightly to Hib porin. To generate large amounts of Hib porin devoid of LOS, the ompP2 gene was cloned into an expression vector of Bacillus subtilis. Recombinant Hib porin was produced in large quantities and it aggregated into inclusion bodies. Under denaturing conditions, recombinant porin was purified to homogeneity and subsequently refolded. To assess the fidelity of refolding of recombinant porin, four criteria were used: spectroscopic properties, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by monoclonal antibody Hb-2. We conclude that in the absence of LOS, recombinant porin was refolded into a functional form, its structure closely resembling the native state.

Although leukotrienes (LTs) are implicated in the pathogenesis of asthma, it is still unclear which types of cells are of primary importance in their formation in asthmatic lungs. To elucidate the mechanism for the increased LT formation in asthma, we investigated the pulmonary cellular infiltration and LT synthesis by dispersed lung cells from BN rats following allergen challenge. / To enable us to do these studies we have developed improved HPLC methods for the analysis of complex mixtures of eicosanoids in biological samples using binary gradients containing trifluoroacetic acid. Samples containing PGB$ sb2$ (the internal standard), cysteinyl-LTs, LTB$ sb4,$ hydroxyeicosatetraenoic acids (HETEs) and the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHTrE) can be analyzed in as little as 20 min. Mixtures which also contain more polar eicosanoids such as lipoxins and omega-oxidation products of LTB$ sb4$ can be analyzed in 40 min. / Ovalbumin (OVA) challenge of sensitized BN rats resulted in a significant influx of neutrophils into the lungs and a significant increase in the synthesis of 5-lipoxygenase products, in particular LTB$ sb4$, by lung cells 6 h after challenge. However, there was little change in the production of cysteinyl-LTs compared with saline challenge. There was a significant eosinophilic infiltration in the lungs 24 h after OVA challenge, but cysteinyl-LT production by lung cells was unaltered at this time suggesting that eosinophils from BN rats are unlikely to be a major site for the formation of these compounds. This was confirmed in experiments with partially purified eosinophils obtained from Sephadex-treated rats. In contrast, cysteinyl-LTs were synthesized in appreciable amounts by alveolar macrophages from BN rats. Administration of rabbit anti-rat PMNL serum abolished the influx of neutrophils into the lungs and appeared to reduce the LTB$ sb4$ synthesis by lung cells. There was a positive correlation between the numbers of neutrophils in the lung and LTB$ sb4$ production by lung cells. In contrast, macrophages were positively associated with cysteinyl-LT production. Exogenous LTB$ sb4$ and 5-oxo-ETE induced infiltration of eosinophils into the lungs and therefore may be responsible for the late phase eosinophilic infiltration in BN rat lungs following allergen challenge.

With the improvement of working conditions in asbestos industries and the recognition that asbestos exposure is widespread, the asbestos controversy has shifted in the 1980s to the general environment. To assess lung cancer risks due to environmental asbestos exposure, risks in past asbestos workers have been extrapolated linearly to exposures 100,000 times smaller than historical occupational levels. Despite their enormous health and economic impact, such risk assessments have not been validated to this day. / This is the first study to compare the risk of lung cancer in a population non-occupationally exposed to asbestos with those predicted by the U.S. Environmental Protection Agency's (EPA) exposure-effect model. The study was carried out among the female population of Quebec's two chrysotile-mining agglomerations, where asbestos pollution has been visible commonly from 1882 to 1975. Six small exposure studies were conducted. These studies were synthesized by a panel of 5 international experts to estimate the historical levels of asbestos in the three main mining towns as well as the asbestos pollution brought home on the clothes of workers. These estimates were combined with a survey of lifetime neighbourhood and household exposures of female residents to assess the cumulative exposure of the females aged 30+ who resided in the mining agglomerations between 1970 and 1989. The average was 35 continuous fiber-years/mL. On the basis of the equivalent occupational exposure, the EPA model predicted a lung cancer relative risk of 2.47. Mortality of the female population of the asbestos-mining agglomerations aged 30+ was compared over the 1970-1989 period to that of 60 comparable agglomerations of Quebec. The lung cancer SMR was 0.99 (95%CI: 0.78-1.26) and the SPMR was 1.10 (95%CI: 0.88-1.38). Although the EPA model predicted 105 excess lung cancer deaths based on SMRs, none were observed in this population; based on SPMRs, an excess of 95 excess deaths was predicted, but only 6.5 observed. The EPA risk assessment on asbestos greatly overestimated the risk of lung cancer attributed to environmental asbestos exposure in this population.