Multiple sclerosis (MS) is considered an immune-mediated disorder of the central nervous system (CNS) characterized by multifocal areas of inflammation and demyelination. Lesion formation depends on migration of lymphocytes from the systemic compartment into the CNS through the blood-brain barrier. We have defined molecular and functional properties of human brain derived microvascular endothelial cells and of T lymphocytes that regulate the migration process and analysed how changes in these properties, in response to inflammatory conditions, contribute to the pathogenesis of multiple sclerosis. Rates of migration of T lymphocytes derived from patients with MS across either a fibronectin and/or endothelial cell barrier in vitro were increased compared to cells obtained from healthy controls. In the MS patients, migration rate correlated with disease activity, defined by clinical and MRI criteria and with IFN gamma production by T cells. Migration was reduced in patients receiving current therapies for MS. Adhesion molecules, chemokines and matrix metalloproteinases were all found to regulate the migration of MS derived lymphocytes through proteins of the basement membrane and human brain endothelial cells. We further demonstrated that the migratory properties of lymphocytes and both permeability and chemokine production by human brain endothelial cells, can be regulated via signalling through the specific kinin B1 receptor that is up-regulated on lymphocytes and human brain endothelial cells during inflammation, providing a potential novel therapeutic approach to modulate lymphocyte-endothelial cell interactions.

Heat shock or stress proteins (HSPs), are a large family of phylogenetically conserved molecules that can be constitutively expressed at high levels under normal physiological conditions. These proteins are selectively synthesized following metabolic and environmental insult. This study was designed to determine whether cognate and/or inducible HSP production are altered in CD4-expressing lymphocytic cell lines concomitant with acute and chronic human immunodeficiency virus type 1 (HIV-l) infection. Our findings indicate that in CEM.NKR cells, HSP27 production is selectively altered at early stages of acute HIV-infection (6-24h post-infection), subsequent to virus internalization but prior to synthesis of viral progeny. Levels of HSP27 induction were viral dose-dependent and were not accompanied by any alterations in cellular proliferation. In contrast, acute HIV-infection was not associated with significant quantitative changes in the constitutive expression of HSP60, HSP70, or HSP90. Nevertheless, a transient, marked induction of select HSP70 subspecies was evident at early stages of infection, finally disappearing by 48-72h. Acute-infection of Jurkat cells resulted in similar patterns of de novo induction of HSP27 and HSP70 isoforms. Uninfected and chronically-infected CEM extracts showed little detectable constitutive HSP27. However, synthesis of select HSP27 and HSP70 homologues in chronically-infected cells was observed following exposure to a mild heat shock and doses of TNF$ alpha$. Similar HSP70 homologues arose in chronically-infected cells treated with heat-shock and TNF$ alpha$. These findings indicate that HSP pathways are uniquely modulated in CD4+ cells as a consequence of acute and chronic HIV-1 infection.

The research work presented in this thesis involved the examination of several fundamental questions concerning the role of oral anaerobic spirochetes (OAS) in the etiology of periodontal disease (PD). OAS are unusual, helical, Gram-negative bacteria that are considered putative periodontopathogens due to numerical association with diseased sites and the enzymatic arsenal available to OAS that appears consistent with disease symptoms. T. denticola is the most commonly isolated OAS from periodontal pockets and as such is the focus of most investigations into the role of OAS in PD. / As free iron is severely limited in humans the means by which OAS may obtain sufficient iron to prosper in the sub-gingiva was examined. The resultant model suggests T. denticola obtains iron through the $ beta$-hemolysis of erythrocytes and the binding of liberated hemin by a 47-kDa outer membrane sheath (OMS) protein. The kinetics of the ligand-receptor interaction are presented and the receptor has been purified to apparent homogeneity from T. denticola. $ sp3$H-labeled hemin is not transported into the cell. Evidence is presented to show that T. denticola produces an iron reductase, which may facilitate the transport of ferrous iron across biological membranes. It is also shown that T. denticola (Td), T. vincentii (Tv) and T. socranskii (Ts) do not produce siderophores. In growth assays, under conditions of iron-limitation, T. denticola may use inorganic iron, a source unlikely to be available in vivo. / Hyaluronidase (Hase) activity is elevated in the gingival crevice during episodes of disease. Hase, when injected into the periodontal cavity under experimental conditions has been shown to result in connective tissue degradation. It is also known that T. pallidum, the agent of syphilis, produces a Hase that is critical to pathogenesis. Evidence is presented herein to show that Td, Tv and Ts all produce a hyauronoglucosaminidase (HGase). The identity and specificity of the Td HGase is confirmed through the use of enzyme inhibitors and activators, by electron microscope observations of the enzyme using the Hase inhibitor gold sodium thiomalate and anti-Apis mellifera venom antibodies and examination of the purity of the HA substrate using other polysaccharide degrading enzymes. As the HGase of these OAS would not migrate through a substrate-SDS PAGE system, we have employed hyaluronate (HA)- and chondroitin sulfate (CS)-absorbed nitrocellulose membranes to visualize HGase activity. The 59 kDa HGase of Td has been purified to apparent homogeneity through the conjugation of HA and CS to Affigel 701 beads. / The last subject to be addressed by this thesis pertains to the ultrastructure of oral spirochetes. Using the copper-containing dye, Alcian blue, we have shown that T. denticola produces an exopolysaccharide layer, in an electron microscopy investigation. The development of a stain for dark-field microscopy has simplified the observation of this layer. The exopolysaccharide layer may have relevance to the evasion of phagocytosis, to protection against colicins, immunoglobulins and bacteriophages, to adherence and perhaps to the immunogenicity of OAS inhabiting the sub-gingiva.

Leukocyte recruitment into tissues in response to infection or injury is a crucial event for the elimination of pathogens to protect the host. However, when leukocytes invade the central nervous system (CNS) and neuromflammatory disorders result, neurological function may be compromised. Infiltration of the CNS, predominantly by T cells and macrophages, characterizes Multiple Sclerosis and its animal counterpart, Experimental Autoimmune Encephalomyelitis (EAE). / Autoreactive T cells that initiate EAE produce Th1 cytokines (e.g., IFNgamma, TNFalpha). Nevertheless, previous studies also indicated an unnecessary or even protective role for IFNgamma in EAE. I have identified a novel role for IFNgamma in my studies using IFNgamma- or IFNgammaR-knockout mice. IFNgamma promotes the expression of the chemokines RANTES, MIP-1alpha, and MCP-1, which recruit mononuclear cells in the CNS to induce a non-lethal remitting EAE. Without IFNgamma, the chemokines MIP-2 and TCA-3, and polymorphonuclear leukocytes prevail, producing an unusually lethal EAE. MIP-1alpha is, however, dispensable in recruiting mononuclear cells, as EAE could still be induced in mice deficient in MIP-1alpha or its CCRS receptor. / To examine how much T cells depend on the cooperation with macrophages in the CNS to induce EAE, selective depletion of peripheral macrophages in mice was achieved by intravenous administration of clodronate-loaded liposomes. Treated mice showed no clinical signs of EAE following adoptive transfer of myelin-reactive T cells, but an altered distribution of leukocytes. These leukocytes were confined within the perivascular or meningeal space, not invading the CNS parenchyma. Levels of TNFalpha and inducible nitric oxide synthase (iNOS) in the CNS were reduced in these asymptomatic macrophage-depleted mice compared to untreated mice with EAE. In these asymptomatic mice, NOS expression was restricted to parenchymal astrocytes. In mice with EAE, however, both macrophages/microglia and astrocytes in infiltrates expressed NOS. Surprisingly, some astrocytes that were distant from infiltrates also expressed NOS, thus suggesting that astrocytes may modulate leukocyte infiltration via release of NO through their foot processes in the blood-brain barrier. Collectively, my data propose a model of a dynamic network in which the interplay among cytokines, chemokines and nitric oxide, may determine the magnitude, the composition, or the resolution of inflammatory infiltrates, as well as the clinical outcome of EAE.

The dystrophin associated protein (DAP) complex has been implicated in such basic physiological processes as the maintenance of cellular homeostasis, the assembly of basement membranes (BM), and synaptogenesis. The dystroglycans, alpha and beta, constitute the functional core of the DAP complex since they establish the transmembrane link between dystrophin, whose mutations lead to Duchenne (DMD) or Becker (BMD) muscular dystrophy, and the extracellular matrix (ECM). The alpha and beta subunits of dystroglycan result from posttranslational processing of a single propeptide encoded by DAG1. No clinical cases or animal models have been identified with spontaneous mutations in that gene. Therefore, to gain insight to the function of dystroglycans I have used gene targeting to disrupt the coding sequence of Dag1 in murine embryonic stem (ES) cells. We find that dystroglycans are critical in the early stages of development and that a null mutation in Dag1 results in embryonic lethality at embryonic day (E) 6.5 because of a disruption in Reichert's BM. In culture however, the absence of dystroglycan in ES cells targeted for both Dag1 alleles does not obviously hinder their developmental potential. Consequently, I reasoned that it might be possible to circumvent the early lethality observed in 'classical' knockout mice, by injecting Dag1-null ES cells into wild-type blastocysts to generate chimeric mice only partially devoid of dystroglycan. Several chimeric mice developed to maturity and Dag1 -null ES cells were found to contribute extensively to the hindlimb musculature thus allowing the analysis of dystroglycan depleted muscles. These muscles are severely dystrophic, have low levels of dystrophin and a disrupted residual DAP complex, but have apparently normal BMs. Chimeric muscles also have disrupted neuromuscular junctions. In culture, myotubes derived from Dag1-null ES cells form clusters of acetylcholine receptors (AChRs) but these occupy a surface area three time

The purpose of this study was to provide evidence for a field defect around colorectal carcinomas using c-fos, mutant p53 and statin markers. Tissue from ten colorectal carcinomas and mucosa at 1, 5 and 10 cm from the primary lesion was obtained from surgical specimens and frozen in liquid nitrogen. Detergent-extracted protein was separated by electrophoresis through polyacrylamide gells and western blots performed using monoclonal antibodies against c-fos, mutant p53 and statin. Expression of c-fos within the carcinoma was increased relative to its expression at 1 cm, which was increased relative to 5 or 10 cm. The reverse results were obtained for statin with the lowest expression detected in the carcinoma, intermediate expression at 1 cm, and highest values at 5 and 10 cm. Increased mutant p53 expression was detected only within the carcinoma. These results indicate that c-fos gain and statin loss may occur before p53 mutation and be initial steps in oncogenesis.

Concern exists that individuals with systemic lupus erythematosus (SLE) have increased susceptibility to cancer compared with the general population. My thesis contains five chapters, each presenting a different perspective on the issue of cancer in SLE. / Although past literature has suggested an increased risk of cancer in SLE, conclusions from earlier studies were not uniform. I review this earlier data in the first chapter of my thesis. / The absence of adequate data regarding malignancy risk in SLE meant that a large, multicentre effort was needed. We have recently completed this multi-centre cohort study, comparing cancer risk in SLE relative to the general population. In the second chapter I present my analyses of these data, which confirm an increased risk of cancer in SLE. The risk is particularly evident for non-Hodgkin's lymphoma (NHL), where an almost four-fold increased risk is estimated. / A potential bias which has been invoked as a possible explanation for the associations between cancer and other chronic disease exposures has been variously called "misclassification", "detection" or "surveillance" bias. If this bias, related to a potential for greater scrutiny for cancer in SLE patients, does exist, one could expect that cancers in SLE patients are diagnosed at earlier stages than in the general population. I examine this in the third chapter, presenting my work that does not support the presence of "surveillance" bias in the results from the multi-centre SLE cohort study. / In the fourth chapter, I describe the demographic factors, subtypes, and survival of the NHL cases that arose in the multicentre SLE cohort sample. The data suggest that aggressive NHL subtypes and poor outcome are common in SLE. / Though the pathogenesis of cancer in SLE is unknown, one theory is that exposure to immunosuppressive medications is a factor. Although definitive evidence is not available, I present, in the final chapter, my findings within the Montreal General Hospital SLE cohort, where immunosuppressive exposure was associated with abnormalities on cervical cancer screening (Pap) tests. / In summary, our work demonstrates an increased risk of cancer in SLE; this is not likely due to surveillance bias. Immunosuppressive exposure may be associated with abnormal Pap tests; further work will determine whether immunosuppressives confer risk for other neoplastic events in SLE, particularly NHL.

Cardiac action potential characteristics are known to vary in different species, but also in the different regions of the heart within a given species and in cardiovascular disease. The heterologous expression of voltage-gated ion currents is believed to underlie these differences. The purpose of this thesis is to elucidate the molecular mechanisms which may underlie some of the observed current changes in different species, as well as regions and diseases of the heart. / Here, we describe the variable dependence on repolarizing K+ currents in different species as being the result of the lack of Ito subunits in guinea pig heart with a greater expression of IK subunits, while rabbits express all hypothesized Ito subunits, but express IK subunits at low levels. Humans are found to lie in between these two species in terms of the expression of these voltage-gated K+ channel subunits. The specialized function of certain regions of the heart, such as the ventricles and the SAN, have been attributed to the heterologous expression of Ito and the pacemaker current (I f) respectively. Here were demonstrate that both Kv4.3 and KchIP2 gradients underlie an observed Ito transmural gradient and contribute to the dispersion of repolarization, while a greater expression of HCN2 and HCN4 subunits in the SAN compared to the right atrium account for the larger I f current in this region. Cardiovascular diseases such as congestive heart failure (CHF) have been associated with ion channel remodelling. Here, we report the finding of changes in Nav1.5, Kv4.3, HCN2 and HCN4 expression which may underlie some of the electrophysiological changes associated with this disease. Furthermore, we characterise a genetic polymorphism which is associated with another disease, atrial fibrillation. / The heterologous expression of voltage-gated ion channel subunits may account for many of the species-, region- and disease-specific differences which have been observed in the heart. Such heterogeneity contributes to the proper functioning of the heart under normal conditions, but may also contribute to the pathogenesis of cardiovascular disease.

The biochemical characterization of the pro-opiomelanocortin (POMC) products, isolated and purified from a human anterior lobe pituitary adenoma associated with Cushing's Syndrome, illustrates the processing of this prohormone. In the tumor, 87% of the acidic joining peptide (AJP) is found as a dimer rather than as a monomer, the prevalent form found in normal pituitary tissue. The corticotropin (ACTH) purified and characterized from the tumor was found not to be phosphorylated. In contrast, human ACTH of normal pituitary origin is found to be 30% phosphorylated at the serine residue at position 31. No evidence of cleavage at residues 49-50 to produce $ tau sb3$-MSH and the 1-49 fragment was found. / ACTH was purified from extracts of guinea-pig anterior pituitaries and characterized in terms of its amino acid composition and molecular weight using IS-MS and HPLC. Guinea-pig ACTH was found to have a similar activity to that of human ACTH with respect to the maximal steroid output of corticosterone and aldosterone. However, it proved to be slightly more potent in terms of the concentration which elicited half-maximal steroid secretion. Under the assay conditions used, guinea-pig ACTH does not seem to a superagonist as suggested by a previous study. Combining amino acid compositions, mass spectrometric data, and the recent determination of the cDNA sequence for guinea-pig ACTH, the identification of various purified biosynthetic derivatives of guinea-pig POMC was facilitated. / Joining peptide, a major product of POMC processing, was found in extracts of both anterior and neurointermediate lobes. The purified peptide corresponded exactly in amino acid composition and mass to the predicted structure established by the CDNA sequence. Also, by using ion-spray mass spectrometry, post-translational modifications of other products of intermediate lobe processing were observed. N- and O-acetylation of $ alpha$-melanotrophin, partial O-phosphorylation of corticotrophin-like intermediate lobe peptide and carboxyl-terminal amidation of $ beta$-melanotrophin were identified. (Abstract shortened by UMI.)

Electroporation refers to a technique that makes use of electrical pulses to create transient and reversible pores into cellular membranes which allows the passage of drugs and macromolecules, such as plasmid DNA, into the cell's interior. It is a widely used laboratory procedure for the transfection of cultured mammalian cells, plant cells and bacteria. Only recently has in vivo electroporation been used for the transfer of genetic material into living tissue, such as muscle, hepatocytes, and tumors of experimental animals. Using a luciferase assay, we have shown that electroporation-mediated luciferase gene transfer into solid, subcutaneous tumors in mice resulted in a 103 fold increase in tumor luciferase activity compared to naked DNA injections. Using this technique, we succeeded in inhibiting the growth of LLC tumors in mice by delivering plasmid DNA encoding mouse Endostatin and mouse single-chain IL-12 directly into the tumor site. Lastly, in vivo electroporation was used to enhance DNA vaccination with the tumor associated antigen CEA and IL-12 gene adjuvant, which protected mice from a tumor challenge with LLC/CEA+ tumor cells. Our results suggest that in vivo electroporation is an efficient nonviral, gene transfer vehicle that can be used in the fields of antiangiogenic cancer gene therapy as well as cancer immunotherapy.