<p>Conclusions about the effects of pregnancy in women - how they feel about being pregnant and their attitudes to the unborn child - have been based almost exclusively upon observations of primigravidas. It is claimed that a satisfying and/or more stressful than later pregnancies. The research for this thesis suggests that some of these claims are mistaken.</p> <p>This thesis examines the similarities and the differences between a group of primigravidas and a group of second pregnancy multigravidas on a range of maternal attitudes during pregnancy. The predictions were based on findings from a pilot study conducted at McMaster University Medical Centre. An interview was designed to elicit the women's thoughts and feelings about their expected infant and about themselves as mothers.</p> <p>Primigravidas and multigravidas were found to be equally positive about the coming baby and equally anxious about their capacities as mothers. The primigravidas reported significantly more anxiety about the welfare of the expected infant and the multigravidas reported significantly more conflict and negative feeling. The common and unique features of a first and a second pregnancy are discussed. The findings suggest that new adaptations and family real realignments accompany the birth of each child.</p> <p>A third sample of women was examined using the same measures. Comparisons were made between a group of multigravidas who had lost an infant by stillbirth or neonatal death and a group of multigravidas without a history of infant loss. Women who had previously lost an infant were found to be still mourning the death of the first infant and less invested in a relationship with the expected infant. The thesis discusses the effects of infant loss upon women and makes recommendations for the clinical management of bereaved mothers.</p> / Doctor of Philosophy (PhD)

<p>Breathlessness may be defined as the conscious awareness of respiratory muscle effort. As with any skeletal muscle it is to be expected that the sense of effort increases as the pressure generated by this muscle increases as well as the velocity and extent of shortening. The purpose of this study was; 1. to quantify the intensity of breathlessness during exercise and respiratory loading; 2. to isolate the contributions of inspiratory muscle pressure to breathlessness; 3. to see if extent of shortening, velocity of shortening, frequency (fb), and duty cycle (Ti/Ttot) contribute to the intensity of breathlessness independently. The intensity of inspiratory muscle pressure was quantified by measurement of mouth pressure (Pm) as well as the estimated esophageal pressure (Pes), the extent of shortening by tidal volume (Vt), and the velocity of shortening by inspiratory flow (Vi). Six normal subjects underwent eight incremental (100 kpm/min/min) exercise tests on a cycle ergometer to maximum capacity. The first and last test were unloaded and the intervening tests were performed with external added resistances and elastances presented in random order. The resistances and elastances were selected to provide a wide range inspiratory pressures, tidal volumes, and flows. The inspiratory resistive loads (33, 57, 73 cm H2 O/1/s) were used mainly to vary the flow (functional velocity of shortening of inspiratory muscles). The inspiratory elastic loads (21, 41, 52 cm H2O/1) were used mainly to vary the tidal volume (functional extent of shortening). At rest and at the end of each min during exercise the subjects estimated the intensity of breathlessness (Y) by selecting a number ranging from 0-10 (Borg psychophysical scale), 0 indicating no appreciable breathlessness and 10 the maximum tolerable sensation.</p> <p>When the velocity was altered (resistive loading study) breathlessness was significantly related to inspiratory pressure (p<0.0001), peak inspiratory flow (p<0.0001), frequency of breathing (p<0.01) and duty cycle (p<0.01). When the extent of shortening was altered (elastic loading study) breathlessness was significantly related to inspiratory pressure (p<0.001), tidal volume (p<0.001), and frequency of breathing (p<0.001).</p> <p>The results indicated that the perceived magnitude of breathlessness is closely related to the pressure generated by the inspiratory muscles and the shortening pattern of these muscles as reflected in Vt, Vi, Fb, and Ti/Ttot. The results also indicated that the contribution of these factors to the intensity of breathlessness differs quantitatively between loaded and unloaded breathing. Thus, in normal unloaded breathing the velocity and degree of shortening are important factors contributing to breathlessness during exercise; with resistive loading the inspiratory pressure, the velocity, and the duty cycle are important; with elastic loading the inspiratory pressure, the extent of shortening, and the frequency are important.</p> <p>The major contributions of these studies were in quantifying the intensity of breathlessness, and defining both the factors contributing to breathlessness and the relative importance of each.</p> / Doctor of Philosophy (PhD)

<p>Despite recent interest in developing novel microparticle (MP)-based antigen delivery systems for mucosal vaccination, existing MP formulations possess a number of liabilities potentially precluding their widespread use. Nevertheless, there is sufficient evidence to suggest that if immunogenically stable vaccine material can be incorporated into biocompatible/biodegradable MPs and delivered to various mucosal surfaces, this may be an effective way to elicit protective systemic and mucosal immunity. To overcome difficulties associated with existing MP formulations, the present work describes the development of a novel polymergrafted starch MP system which is capable of entrapping a wide variety of soluble antigens under mild conditions and which will elicit efficacious immune responses following intragastric (i.g.) or intranasal (i.n.) administration. The novel MP delivery technology was developed using starch, a well-studied, biologically acceptable and non-toxic material when given orally or parenterally. Antigen-containing starch MP were subsequently grafted with a hydrophobic silicone [3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane, TS-PDMS] which, it was hypothesized, would protect microentrapped antigen from the deleterious environment found in the gastrointestinal (GI) tract, facilitate MP uptake by M cells overlying mucosae-associated lymphoid tissues (MALT) and/or act as an adjuvant or immunopotentiator. Moreover, following selective transport into mucosal inductive sites, antigen-containing MPs should stimulate the generation of robust antigen-specific disseminating mucosal and circulating humoral immune responses. In the work reported here, it was demonstrated that antigen-containing TS-PDMS-grafted MPs could be fabricated under mild conditions, allowing for the successful entrapment of a variety of protein and peptide antigens without any demonstrable loss in immunogenicity. Furthermore, it demonstrated that under acidic conditions, protein release from MPs was retarded when MPs were grafted with TS-PDMS. Although protein release from TS-PDMS-grafted MPs compared to ungrafted MPs was hindered, microentrapped protein was still released from TS-PDMS-grafted MPs, thus demonstrating that the MP composition allowed for entrapped proteins to readily learn from the MP matrix. Overall, these observations suggested that TS-PDMS-grafted MPs could serve as an efficacious MP-based mucosal vaccine delivery system. To be considered an attractive alternative to current mucosal MP vaccine delivery vehicles, TS-PDMS-grafted MPs should incite both local mucosal and systemic humoral immunity following mucosal administration of low doses of microentrapped antigen. The studies in this report clearly demonstrated that oral immunization with very low doses of TS-PDMS-grafted MPs simulated both systemic and mucosal humoral immune responses. Indeed, the adjuvanticity of TS-PDMS-grafted MPs seems to have arisen via a unique physicochemical relationship occurring between protein antigen and silicone in the starch matrix. This led to a predominantly Th2-type immune response following oral MP administration of relatively small amounts of microentrapped antigen. Furthermore, serum antibody titres were augmented after an oral or systemic boost, suggesting that the initial immunization protocol stimulated serum antibody memory capability, an advantage when developing successful mucosal immunization strategies. Surprisingly, systemic antigen challenge failed to boost antigen-specific sera igA titres following i.g. immunization with microentrapped or soluble antigen. These results suggest Peyer's patch (PP)-stimulated igA lymphocytes migrate to mucosal lymphoid compartments following i.g. MP administration, while antigen-specific PP-stimulated IgC plasmacytes have the propensity to migrate to both mucosal and systemic lymphoid compartments. In addition to stimulating specific serum antibody responses, i.e. immunization with TS-PDMS-grafted or ungrafted MPs resulted in specific sIgA responses in the gut; this is in contrast to soluble antigen which was incapable of inciting specific intestinal immunity following i.g. administration. Thus, compared to soluble antigen, TS-PDMS-grafted MPs have immunopotentiating activity when delivered orally. The studies outlined in this work strongly suggest that i.g. administration of TS-PDMS-grafted MPs stimulated mucosal immunity via PP. Following i.g. immunization with a low dose of antigen-containing MP, but not soluble antigen, specific proliferation of PP cells was observed. Lymphocyte proliferation was subsequently observed in mesenteric lymph node (MLN) and splenic tissue. In contrast, antigen-specific lymphocyte proliferation was not observed in gut lamina propria (LP) lymphocytes following i.g. immunization with MPs, thus suggesting that MP-induced immunity was incited by MP uptake and processing solely by PP. It was shown also that i.n. immunization with low doses of microentrapped, but not soluble, antigen evoked robust circulating specific IgG responses, indicating that TS-PDMS-grafted MPs could enhance the immunogenicity of an i.n. administered soluble antigen. Indeed, i.n. immunization with microentrapped protein stimulated greater levels of specific sera IgG than was observed after i.g. MP administration with equal amounts of microentrapped antigen. However, unlike i.g. TS-PDMS-grafted MP immunization, antigen specific IgA was not detected in local mucosal secretions or sera following i.n. immunization with microentrapped antigen, thereby suggesting that i.n. immunization expresses unique immune responses compared to those evoked after comparable i.g. immunization protocols. Although that nasal-associated lymphoid tissue (NALT) is considered to be the equivalent of Waldeyer's ring in humans, the exact process of generating immune responses when NALT is exposed to antigen is not clear. The present work describes the development of a novel, rapid and precide method for isolating NALT from mice to study its immune function and cell populations. Following the development of this precise NALT isolation technique, the pathway of i.n. administered TS-PDMS-grafted MPs was examined. It was demonstrated that i.n. immunization with low doses of microentrapped, but not soluble protein, evoked robust circulating IgG responses via NALT cell activation. Numerous antigen-specific spot-forming cells (SFCs) were observed in the NALT and later the posterior cervical lymph nodes (pCLN) and spleen (SPL), confirming the selective drainage of NALT cells exclusively to the pCLN following i.n. administration of a particulate antigen. Although B cell activity was observed in the pCLN following i.n. MP administration, no specific IgA was detected in any lymphoid tissue examined or in nasal secretions following i.n. immunization with TS-PDMS-grafted MPs. This suggested that either pCLN in mice are not intermediate in evoking sIgA responses in the nasopharynx or that TS-PDMS-grafted MPs are incapable of stimulating this arm of the murine immune system. However, since it was previously demonstrated that i.g. immunization with comparable doses of TS-PDMS-grafted MPs evoked intestinal IgA responses, the inability to detect antigen-specific IgA in nasal secretions probably reflects differences between murine NALT and PP and not a unique inability of TS-PDMS-grafted MPs to evoke secretory immunity in the nose. Thus, the present work describes the successful development of a novel polymer-grafted starch MP system which is capable of entrapping a wide variety of soluble antigens under mild conditions and which can elicit efficacious immune responses both orally and nasally, via MALT activation.</p> / Doctor of Philosophy (PhD)

<p>Classical force-velocity studies by A. V. Hill demonstrated that there was an optimal velocity for maximal power output of isolated muscles and human movements. Thus to study muscle performance during maximal dynamic exercise it is important to measure mechanical power output at several constant velocities of movement. At the start of this work no instruments were available to measure maximal power during isokinetic movements over a wide range of velocities. For this reason a cycle ergometer (CVE) was developed which restricted the crank velocities to chosen upper limits, despite maximal efforts by the subject.</p> <p>Measurements were obtained in male subjects of maximal peak torque generated over 81% of the functional range. There was a consistent inverse linear relationship between peak torque, and crank velocity, and the results were reproducible from day to day. Considerable inter-subject variability in peak torque was accounted for only partly by differences in thigh muscle volume. Maximal peak power occurred at various crank velocities ranging from 120 to 160 rpm; differences in muscle fibre types may have contributed to the variation observed. Maximal power occurred when the force equalled 0.3 to 0.4 of the predicted maximal isometric tension, in agreement with Hill's studies.</p> <p>Torque, work and power were also measured during 30 s of maximal effort at 60, 100 and 140 rpm. Increases in crank velocity were associated with both a higher initial power, and a greater rate and extent of decline in power, but total work was similar. The greater decline at faster velocities may reflect differences in energy metabolism, or motor unit activation. In addition to defining the effects of velocity on maximal power output in healthy young subjects, the studies showed the CVE to be a sensitive, reliable instrument, with potential applications to the assessment of human muscle function in health and disease.</p> / Doctor of Philosophy (PhD)

<p>Many tumors express antigens that are capable of being recognized by effector cells of the immune system. However, these tumors persist in immunocompetent hosts. The mechanism by which immune effector cells are rendered unresponsive to these antigens is unknown. Previous data from our laboratory demonstrated the use of adenoviral vectors constructed to express single cytokines (IL-2 and IL-12) in mediating regression of tumors in a mouse mammary adenocarcinoma model. The ability of these molecules to enhance the immune response to effectively regress these tumors was hampered by toxicities associated with these factors. To over come this toxicity and enhance the efficacy of IL-2, double recombinant adenoviral vectors expressing the co-stimulatory molecules B7-1 or B7-2 with IL-2 were constructed to allow for the expression of both immuno-modulatory molecules from the same cell. An alternative approach to enhancing the efficacy of these factors involved the use of double recombinant adenoviral vectors expressing combinations of the chemokine lymphotactin with IL-2 and IL-12. Mammary tumor cells from transgenic mice expressing polyoma middle T (PyMT) or an oncogenic form of the proto-oncogene Neu (Neu 8142) were transplated into syngeneic FVB/N recipients to establish the subcutaneous tumor models. Intra-tumoral injection of adenoviral vectors constructed to express murine B7-1 or B7-2 and IL-2 resulted in regression of PyMT or Neu (8142) tumor bearing mice, superior to that observed for the single cytokine or co-stimulatory molecule expressing vectors. Cured mice were shown to have generated systemic immunity to a subsequent challenge with fresh tumor cells and tumor specific cytotoxic T lymphocyte activity could be detected. Intra-tumoral injection of adenoviral vectors constructed to express lymphotactin IL-2 or IL-12 demonstrated complete regression in a small number of PyMT tumor bearing mice. None of these single cytokine or chemokine expressing vectors was capable of inducing regression of Neu (8142) tumors. In contrast, administration of vectors expressing either a combination of lymphotactin and IL-2 or lymphotactin and IL-12 resulted in an increased incidence of complete regression in both tumor models. All regressed mice demonstrated systemic protection and anti-PyMT CTL. The activity of these vectors in the Neu tumor model is different and suggests some variance in the immunogenicity of the two tumor models. These results demonstrate the ability of double recombinant vectors expressing the co-stimulatory molecules (B7-1 or B7-2) or lymphotactin in combination with IL-2 or IL-12 to enhance the anti-tumor effects of either cytokine alone. These vectors demonstrated no associated toxicities, due to the reduced levels of cytokine that are needed to maximally co-operate with the co-stimulatory or chemokine molecules. Development of this type of vector system will lead to much improved clinical protocols for cancer therapy.</p> / Doctor of Philosophy (PhD)

<p>The mast cell has been implicated as an initiating cell in the immediate responses to allergen challenge where preformed mediators such as histamine play an important role. However, the role of mast cells is less well understood in severe allergic disorders such as asthma where chronic inflammatory changes are resent. Results presented in this thesis demonstrate that freshly isolated and highly purified rat peritoneal mast cells can release IL-6 without any necessity for histamine release. These observations were determined with the use of bacterial products such as LPS and CT which significantly enhanced IL-6 production in our system.</p> <p>Prostanoids of the E family (PGE₁ and PGE₂) were also used as stimulating agents which may also participate in inflammation. We observed that these prostaglandins selectively enhanced IL-6 production while TNF-α synthesis was significantly inhibited CT was observed to have very similar effects to PGE₁ on mast cells . These findings illustrate the potential role that mast cells may have during chronic inflammation and infectious disease.</p> / Doctor of Philosophy (PhD)

<p>Interleukin 6 (IL-6) is a pleotropic cytokine that has many important physiological roles during inflammation. One function is the regulation of the acute phase response of the liver. In order to understand the role of IL-6 as an inflammatory mediator in the acute phase response, it was necessary to study the effect that this molecule had on the regulation of its own receptors, IL-6 receptor (IL-6R, gp80 or α-receptor), and signal transducing protein, gp 130, both in vivo and in vitro.</p> <p>To investigate the role that IL-6 and corticosterone play in the acute phase response and receptor regulation, we examined serum IL-6 levels, serum corticosterone levels, acute phase protein levels, and the expression of hepatic IL-6R (gp80) and gp 130 mRNA levels, in three different models of acute inflammation. Rats were treated with either Freund's complete adjuvant (FA) via intraperitoneal injection, LPS via intravenous injection, or turpentine via subcutaneous injection. All three models showed increased levels of serum IL-6 activity with LPS-treated rats inducing the quickest and greatest response (>100 ng/ml within 3h). Serum corticosterone levels increased by 3h after all treatments, and serum levels of acute phase proteins were detected within 12-24h. The expression of the IL-6R (gp80) mRNA increased as early as 3h after treatment and mRNA levels began to decline by 6-12 h. The gp 130 mRNA levels increased 2-3 fold within 24h, and the time of maximum increase differed depending on the treatment that the rat received.</p> <p>Another cytokine, Leukemia Inhibitory Factor (LIF), and IL-6 share many functions in vitro, and this redundancy is thought to occur as a result of their two alpha receptors, LIF-R and lL-6R, respectively, interacting with the same signal transducing molecule, gp 130. We examined the increase of LIF-RmRNA levels in the three models of acute inflammation to determine the role of LIF during the acute phase response in comparison to IL-6. We found a maximum 2-3 fold increase in mRNA levels in comparison to controls. Maximum LIF-R mRNA levels varied depending on the type of treatment the rats received.</p> <p>Overall analysis of all mRNA levels studied, showed that maximum IL-6R (gp80), gp 130, LIF-R and Cysteine Proteinase Inhibitor (CPI-an acute phase protein whose expression is regulated by IL-6) mRNA levels peaked at different times depending on the type of acute inflammation induced. Although IL-6R mRNA levels reached maximum levels quickly (3-6h) in all three models of acute inflammation, the maximum induction of gp 130 and LIF-R differed depending on the type of treatment the rats received. In all three acute inflammatory models, IL-6R and LlF-R mRNA leveIs did not peak at the same time for any of the treatments given. This suggests that although IL-6 and LIF may have similar functions in vitro, their role in vivo in inflammation is unique. Staggering of maximum LIF-R and IL-6R expression in the liver may ensure that cells are receptive to continual messages to make acute phase proteins.</p> <p>To further investigate the individual effects of raised corticosterone and 1L-6 on the expression of these receptors, rats were injected with either d examethasone (DEX) or purified recombinant IL-6 (rIL-6) via intraperitoneal injection. Rats injected with rIL-6 showed a dramatic increase in both IL-6R (gp80) and gp 130 mRNA levels, as early as Ih after treatment. Dexamethasone had a significant, but less dramatic effect, on IL-6R (gp80) mRNA levels and no effect on gp 130 message. An acute phase protein response was only seen when rats were injected with rIL-6 and not DEX. Neither rIL-6 or DEX treated rats-had any effect on LIF-R mRNA levels.</p> <p>To continue these studies, we investigated the long term in vivo effects of prolonged exposure to purified rIL-6 on the expression of these hepatic cytokine receptors. Repeated injections of rIL-6 did not cause decreased mRNA levels, instead increased 1L-6R (gp80), gp 130, and CPI mRNA levels (2-3 fold) were seen. Serum CPI protein levels increased gradually over a nine day period from 1.4 mg/ml to 5.8 mg/ml. LIF-R mRNA levels remained unaffected by the repeated injections of rIL-6.</p> <p>The steady-state mRNA levels of the interleukin-6 receptor (IL-6R, gp80) and its signal transducing molecule, gp130, were examined in the rat hepatoma cell line, H-35, stimulated by cytokines IL-6, IL-1, Oncostatin M, and/or DEX. In contrast to our in vivo findings, in vitro DEX seemed to be the major stimulator of IL-6R mRNA expression, whereas IL-6 seemed to have little effect on the expression of its own receptor mRNA levels. However, the presence of other cytokines also influenced the corticosteroid (dexamethasone-DEX) mediated stimulation of IL-6R expression. Oncostatin M, a third cytokine related to IL-6, stimulated lL-6R mRNA levels, and this stimulation was additive with the DEX-mediated stimulation of IL-6R mRNA levels. In contrast, Interleukin 1 (IL-1) inhibited the DEX-mediated stimulation of IL-6R mRNA. At the same time, lL-1 also stimulated the presence of a second smaller mRNA transcript. This mRNA species contained the extracellular domain but lacked both the transmembrane and cytoplasmic domains of the IL-6R, suggesting alternate splicing, possibly coding for a soluble form ofgp80.</p> <p>The expression of the gp130 molecule was not regulated to any major extent in vitro, and cytokines IL-6, Oncostatin M, and IL-1 all stimulated the expression of the signal transducing molecule, gp130, approximately 2 fold.</p> <p>Cysteine Proteinase Inhibitor mRNA and protein levels were elevated by combinations of cytokines: IL-6+DEX, IL-6+IL-1+DEX, OSM, OSM+DEX. However, IL-1 again seemed to inhibit the IL-6+DEX mediated stimulation of CPI mRNA, possibly through inhibition of IL-6R expression and induction of a possible soluble form of the receptor.</p> <p>This study shows that the combination of cytokines and hormones that interact with the hepatocyte and in turn, regulate the expression of these different receptors is very complex. In vitro, we demonstrated that both IL-1 and Oncostatin M are involved in the regulation of the IL-6 receptor complex, and that different combinations of cytokines can affect the complexity and magnitude of the hepatic acute phase response. In vivo, we demonstrated that depending on the type of acute inflammation induced, that a variety of combinations of cytokines and hormones are released, and these in tum regulate the diverse receptors on the hepatocyte, resulting in different acute phase response kinetics. Therefore, even though all of these receptors belong to a family and have similar functions, the regulation of these receptors is unique and may lead to altered cell responses in the different models of inflammation.</p> / Doctor of Philosophy (PhD)

<p>Human somatic cells have a finite lifespan. In contrast, since most cancers evolve through expansions of mutant clones with increasingly more transformed phenotypes, tomour cells may exhaust the proliferative potential of normal cells and possibly acquire an unlimited replicative capacity (immortality). Thus, one essential step in tumourigenesis may be the acquisition of an immortal phenotype. The results presented in this thesis suggest that telomeres, the terminal structures that prevent illegitimate recombination and ensure the proper segregation of chromosomes, and telomerase, the enzyme that elongates telomeres de novo, play critical roles in the process of immortalization of transformed cells both in tissue culture and in vivo.</p> <p>In a tissue culture model of transformation we have shown that telomeres shorten as normal cells divided, as previously reported (Harley et al., 1990), and that, consistent with this observation, the cells lacked detectable levels of telomerase activity. Cells that were driven to divide beyond their normal lifespan by transformation with viral oncogenes did not directly acquire telomerase activity; consequently telemeres continued to shorten until a proliferative crisis, characterized by cell death, was reached. At crisis, chromosome ends contained very little telomeric DNA and appeared to be unstable since the frequency of dicentric chromosomes, aberrations that can be formed by the fusion of chromosome ends, increased. These data suggest that the critically short telomeres detected at crisis may no longer be functional, resulting in genomic instability and potentially cell death. Immortal clones which survived crisis maintained short, but stable telomeres and had telomerase activity. Similarly, malignant cells from the advanced stages of different cancers also had short telomeres and were telomerase positive. Moreover, in one cancer analyzed, the telomeres of malignant cells were found to be stably maintained in vitro and in vivo. These data, although correlative in nature, strongly suggest that, in culture as well as in vivo, telomerase must be activated to counter the lethal loss of telomeric DNA if cells are to become immortal.</p> / Doctor of Philosophy (PhD)

<p>One of the possible contributing factors in the development of hypertension may be an accelerated or premature vascular ageing process, because of some similar structural and functional alterations in the vasculature, including an impaired β-adrenoceptor mediated vascular relaxation and an increase in polyploid smooth muscle cells. The primary objective of this study was to investigate the possible relationship between development of polyploidy and the plasma membrane β-adrenoceptor in cultured smooth muscle cells from the thoracic aortae of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) from 3-4-(prehypertensive), 10-12-(developing hypertensive), and 28-30-weeks (establish hypertensive) of age.</p> <p>The major findings from this study are: (i) similar to the in vivo state, cultured smooth muscle cells from different age-groups of WKY and SHR contain a heterogeneous population of mononucleated and multinucleated cells, as well as diploid and polyploid smooth muscle cells; (ii) the expression of both smooth muscle cell polyploidy and β-adrenoceptor density increases with age in both SHR and WKY, however, this increase was significantly accelerated in SHR as compared to WKY, suggesting an accelerated or premature ageing process may be involved in SHR as compared to WKY; (iii) both SHR and WKY express functional smooth muscle cell β-adrenoceptors but many of the β-adrenoceptors expressed on cultured SHR smooth muscle cells are not coupled to adenylate cyclase; (iv) elevation of intracellular cAMP levels either by agonist activation of β-adrenoceptors or by direct activation of adenylate cyclase in cultured smooth muscle cells from 3-4-week old WKY and SHR and 10-12-week old WKY resulted in an increase in polyploid cells; (v) a β-adrenoceptor antagonist only partially inhibited the isoproterenol-stimulated increase in polyploid smooth muscle cells in both SHR and WKY; and, (vi) the development of polyploid SMC via the β-adrenoceptor-Gs-protein-adenylate cyclase-cAMP pathway is more efficient in cells from WKY compared to SHR.</p> <p>From these findings, I conclude that: (i) the vascular β-adrenoceptor mediated signalling pathway plays a role in the development of polyploid smooth muscle cells; and, (ii) additional intracellular pathways, independent of the β-adrenoceptor-cAMP intracellular mediated signalling pathway, are involved in the development of smooth muscle cell polyploidy.</p> <p>Since it is the resistance arteries (which show no significant increase of polyploid smooth muscle cells with age and/or duration of hypertension) and not the elastic large arteries (aorta) which play a significant role in the development of hypertension, these findings have more relevance to the premature ageing process than to the development of hypertension.</p> / Doctor of Philosophy (PhD)

<p>Cytokines are polypeptide hormones that act nonenzymatically to regulate host cell functions. These glycoproteins make up a fourth class of soluble intercellular signalling molecules that also include neurotransmitters, endocrine hormones and autacoids and are believed to play a central role in tissue remodelling in inflammation, infection, and wound repair. Numerous studies have now implicated cytokines to be of critical importance in host defense, and a more complete understanding of their molecular function is essential. What is also evident is that the majority of biological functions assigned to cytokines have been characterized by in vitro systems.</p> <p>In vivo confirmation of these reported biological functions is required and has been attempted. To date, this has been difficult to attain with the available animal models. While studies in transgenic mice have revealed a number of biological activities, they probably do not reflect normal physiological responses, since tissues chronically exposed to a cytokine throughout development may undergo alterations in its effector phenotype. Administration of recombinant protein is also problematic as repeated injections with large doses of purified recombinant protein are usually required to maintain physiologic concentrations due to the short half life of most cytokines in the circulation.</p> <p>To overcome these problems, we have developed an alternative approach to investigate cytokine function in vivo. This approach, which we have defined as a "pseudo transgenic" animal model, uses recombinant adenovirus vectors containing cytokine genes to deliver and transiently overexpress cytokines in vivo in a tissue-directed manner to normal adult animals. Using this vector approach, cytokine expression can be targeted to a tissue in a way that may mimic more normal physiologic responses. In this study, recombinant adenovirus type 5 vectors capable of expressing the murine cytokines interleukin-5 (IL-5), interleukin-6 (IL·6), and RANTES were constructed to investigate the in vivo effects of these cytokines on immune and inflammatory responses.</p> <p>The first vector constructed, Ad5E3mlL6, contained the murine IL-6 gene incorporated into the E3 region of the viral genome and was used to characterize the capacity of recombinant adenovirus vectors for cytokine expression. This vector was very efficient for cytokine expression both in vitro and in vivo. In addition, using an adenovirus vector containing luciferase as a reporter gene, we demonstrated that expression could be targeted in a highly tissue-specific manner dependent upon the route of administration.</p> <p>Since IL-6 was reported to be the major mediator of the acute phase response and intraperitoneal administration of adenovirus vector primarily targeted cytokine expression to the liver and spleen in Balb/c mice, our initial investigation involved intraperitoneal injection of the Ad5E3mlL6 vector into Balb/c mice. This study confirmed in vivo biological roles for IL-6 as a major mediator of the acute phase response and as a B and T cell proliferation factor.</p> <p>We then analyzed the effects of expression of IL-5 and IL-6, alone and in combination, on humoral immune responses in the mucosal tissue of the lung. Both cytokines, produced by T helper type 2 lymphocytes, are critical to the development and differentiation of B lymphocytes and in particular to IgA antibody production in the mucosa-associated lymphoid tissue (MALT) and bronchus-associated lymphoid tissue (BALT). These studies, using Ad5E3mlL5 (a vector expressing murine IL-5 in the E3 region of the virus) in conjunction with Ad5E3mIL6, provided in vivo support for the roles of IL-5 and IL-6 in inducing lung mucosal immune responses. Co-administration of these two vectors in C57BI/6 mice synergistically induced up to a-fold increases in antigen-specific IgA antibody production in the lung.</p> <p>In addition, we studied the in vivo effects of RANTES, a molecule reported to be chemotactic for monocytes, on lung inflammatory responses. A vector, Ad5E3mRANTES, was constructed which contained the murine RANTES cDNA in the E3 region of the virus. This vector, when intratracheally instilled into Sprague Dawley rats, targeted expression to the mucosal tissue and induced the recruitment of monocytes to the lung within 24 hours. These effects were transient and this expression did not result in detectable lasting pathologic changes to the organ. These in vivo findings on RANTES function are consistent with its proposed function as a potent monocyte chemotactic factor.</p> <p>Finally, we applied this newly developed adenoviral technology to immune modulation in an animal model of breast cancer. Mammary tumor cells from transgenic mice expressing the polyoma middle T antigen under the control of the mouse mammary tumor virus promoter were transplanted into syngeneic mice to establish subcutaneous tumors. These tumors were directly injected with control virus or Ad5E1mlL6A + vector, a replication-deficient vector containing the murine IL-6 gene in the E1 region of the Ad5 genome. We found that localized vector-derived expression of IL-6 could attenuate tumor growth.</p> <p>In conclusion, the results of this thesis study demonstrate the practical use for recombinant adenovirus vectors to aid in the investigation of cytokine function in vivo. This demonstration that vector-derived expression of cytokine is high and can be targeted to specific tissues suggests their use as potential therapeutic agents for the modulation of immune responses in the treatment of cancer and other diseases.</p> / Doctor of Philosophy (PhD)