<p>Alpha-l-protease inhibitor (αlPi) is a plasma glycoprotein which plays a major role in limiting proteolysis during inflammation due to its broad spectrum of inhibitory activity.</p> <p>The purification and partial characterization of αlPi have allowed for its investigation as an acute phase reactant during inflammation.</p> <p>Mouse αlPi is a glycoprotein of molecular weight 53,500 with a half-life of approximately 15.5 hours. On two dimensional immunoelectrophoresis, two variants of αlPi were seen which exhibited immunological non-identity. The two variants showed trypsin binding activity, both were synthesized by hepatocytes, and both behaved as acute phase reactants.</p> <p>The ubiquitous nature of alPi was evident from its presence in a wide variety of body fluids. Using an immunohistochemical stain, the inhibitor was shown to be normally present in the cytoplasm of hepatocytes, islet cells of pancreas and in some villous epithelial cells of the small intestine. The hepatocyte was shown to be the major source of serum αlPi. Alveolar macrophages, shown to contain αlPi histochemically in particular inflammatory states of the lung, synthesized minimal quantities.</p> <p>The acute phase response of αlPi was investigated in four models of inflammation consisting of either a subcutaneous injection of celite or infection with one of three parasites: Nippostrongylus brasiliensis; Trichinella spiralis; and Trypanosoma congolense. In addition, studies have been initiated with other mouse acute phase reactants, namely, complement component C3, serum amyloid P (SAP) and serum amyloid A (SAA).</p> <p>The induction of acute phase protein synthesis during inflammation was characterized by an increase in the number of hepatocytes staining for αlPi. During inflammation the level of hepatocyte staining was shown to correlate with the synthetic output of αlPi. The maximum staining activity for αlPi in hepatocytes preceded the peak increase in serum levels during inflammation. Moreover, there was a characteristic, progressive alteration in the distribution pattern of stained hepatocytes within the liver lobule. These results were consistent with an acute phase mediator perhaps originating from the site of inflammation, gaining access to the liver via the portal circulation causing an induction of acute phase protein synthesis.</p> <p>The synthesis of an acute phase mediator (APM), probably interleukin 1 (ILl), by alveolar macrophages and the in vitro induction by APM of αlPi synthesis by hepatocytes has been demonstrated.</p> <p>It was also shown that αlPi accumulated at the site of inflammation which may account for the apparent lack of increase in serum levels even though there was an increased hepatic output of alPi at that time.</p> <p>Collectively these results demonstrate that in the mouse the induction of synthesis of αlPi during an acute response to inflammation is mediated by an acute phase mediator originating from macrophages at the site of inflammation.</p> <p>The increased synthesis of αlPi has been shown in pivo by a measure of serum levels and immunohistochemical examination of liver tissue as well as in vitro by cultures of isolated hepatocytes.</p> <p>These studies constitute a basic framework upon which mechanisms for the induction and control of synthesis of acute phase proteins can be further explored.</p> / Doctor of Philosophy (PhD)

<p>The arenaviridae are a group of enveloped, negative-stranded RNA viruses. These viruses characteristically produce persistent infections in their natural host, which is how they are maintained in nature. Experimentally-induced arenavirus disease has been found to possess similar pathological features of human disease. In the inbred MHA strain of Syrian hamster, Pichinde virus causes a fatal infection when inoculated intraperitoneally. Investigation of the factors leading to a fatal Pichinde virus disease in Syrian hamsters revealed that susceptibility was genetically determined. Susceptibility of MHA hamsters to a lethal infection of the virus appeared to be related to the presence of a splenic target cell for Pichinde virus replication that was minimally expressed in resistant hamster strains. In addition, during studies on cell-mediated immune responses to Pichinde virus antigens, it was discovered that MHA hamsters survived infection when the virus was given in the footpad. However, unlike the resistant LSH and LVG strain hamsters, the MHA hamsters did not manifest a footpad swelling response.</p> <p>The present studies were initiated to determine some characteristics and possible mechanism(s) underlying the lack of footpad swelling response to a primary inoculation of Pichinde virus in the MHA strain hamster. MHA unresponsiveness was not due to a lack of immune recognition of Pichinde virus antigens, since this strain was shown to produce complement-fixing antibodies to Pichinde virus. Furthermore, footpad-inoculated MHA hamsters were protected against a subsequent intraperitoneal challenge with Pichinde virus.</p> <p>Experiments designed to elucidate the genetic basis for presence or absence of footpad swelling revealed that expression of footpad swelling to Pichinde virus was a dominant trait controlled by a single gene or closely linked genes. Furthermore, the lack of responsiveness in MHA hamsters appeared to be specific for Pichinde virus, since footpad swelling could be elicited in this strain by footpad injection of either vaccinia virus or vesicular stomatitis virus. Histological analysis of the virus-injected footpad showed that footpad swelling was associated with an influx of mononuclear cells at the site of injection. These observations suggested that MHA unresponsiveness could not have been caused by a general defect in effector mechanisms that mediate the footpad swelling response.</p> <p>A search for Pichinde virus-induced suppressor activity was then undertaken. Treatment of MHA hamsters with cyclophosphamide, a drug known to augment delayed hypersenstivity responses by inhibiting suppressor cell activity resulted in a significant footpad swelling response following Pichinde virus injection. Furthermore, transfer of lymphoid cells but not serum from 5 day footpad-inoculated MHA hamsters could specifically suppress footpad swelling to Pichinde virus in the LSH recipient.</p> <p>Aqother parameter of cell-mediated immunity studied was the generation of cytotoxic cells. Pichinde virus primed MHA and LSH hamsters were capable of generating cytotoxic cells, after restimulation in vitro with virus but the cytotoxicity lacked specificity. When spleen cells from 7 day footpad-inoculated MHA hamsters were added to spleen cells from LSH hamsters, the cytotoxic activity of the latter cells was significantly suppressed.</p> <p>These results taken together suggested that the absence of footpad swelling in the MHA strain hamster was caused by the generation of a cell-associated suppressor mechanism that appeared to be induced specifically by Pichinde virus.</p> / Doctor of Philosophy (PhD)

<p>Sexually-transmitted diseases (STDs) are among the most common causes of illness in the world. The annual incidence of STDs is rising, from 250 million in 1990 to 340 million in 2002. Viral infection is a frequent cause of STDs and the mucosal surfaces are the natural sites of transmission of viruses. Both genital and rectal tracts are involved in transmission of viral SIDs. To infect a host, a virus has to penetrate mucosal immunologic barriers, including lumenal immunoglobulin A (lgA) intervention, epithelial defenses, and lamina propria (LP) lymphocyte- ediated protection. The breach of the mucosal immune system can lead to virus spreading to the rest of the body, thereby causing life-threatening disease. Induction of effective mucosal immunity is essential for the host to control local viral infection and prevent SID development. Immune responses are initiated when pathogen-derived antigen (e.g., viral antigen) is encountered and taken by antigen-presenting cells (APes), especially dendritic cells (Des). After antigen processing, Des present immunogenic determinants to and activate naIve lymphocytes in mucosal lymphoid tissues. Activated lymphocytes leave the lymphoid tissue and, via the bloodstream, migrate to the LP. These effector cells exert a series of immune functions, such as cytokine production, cytotoxicity and antibody secretion. Accordingly, the mucosal immune system is divided into the inductive and effector site. Gut-associated lymphoid tissues (GALT) represent the inductive site of the gastrointestinal (GI) system. The GALT within the rectal mucosa mainly consists of iliac lymph nodes (lLN) , Peyer's patch-like aggregated lymphoid follicles (ALF) and isolated lymphoid follicles (ILF). The ILN have been identified as the principal inductive site, whereas the role for mucosally located lymphoid follicles remains poorly understood. To elicit specific mucosal immune responses against virus infection, a viral antigen must be introduced into the mucosal immune system, importantly the inductive site. Efficient antigen delivery ensures the success of inducing effective mucosal immunity. Both the mucus layer and the integral epithelial monolayers form barriers against the passage of proteins and particulate matter across the epithelium. Previous studies demonstrated that these barriers could be overcome by mucus removal with ethanol treatment followed by utilizing viral vectors such as replication-deficient adenoviral vectors (Adv) engineered to encode heterologous antigen genes. Adv can infect a broad range of mammalian cells, including human and ~ouse epithelial cells, and has proved to be efficient in transferring genes to the colonic mucosa. It has been discovered that mucosal immunity can be induced at multiple mucosal sites, but rarely via a systemic route. This phenomenon is largely due to the mechanism of the common mucosal immunologic system (CMIS), within which activated mucosal lymphocytes migrate from one mucosal site (e.g., the upper respiratory tract mucosa) to another (e.g. the genital tract). Thus, the concept of CMIS is used as a guiding principle for mucosal vaccine design. However, emerging results have suggested that distant mucosal immunization is less effective than local immunization and that CMIS might comprise several anatomical-based grouped networks having different lymphocyte homing mechanisms. In this context, the vaginal (local mucosal) immunization regimen might improve local protection against genital STDs. Following the similar logic, rectally-induced immune responses might have potential to combat rectal STD infections.As both genital and rectal mucosae are drained by the ILNs, the putative genito- ectal associated lymphoid tissue, rectal immunization might be an alternative solution to genital immunization, especially to deal with the problem of immunization in the male genitourinary tract. The major goal of this study was to evaluate the effects of Adv-based local mucosal immunization via the rectum of mice in the induction of mucosal immunity against virus infection at the rectal as well as genital mucosa. non-Invasive intrarectal (IR) delivery method (pipetting and Dermabond® following a ethanol enema) for Adv was developed in the present study to provide better gene transfer for induction of mucosal immune responses. The first approach was to reinvestigate gene transfer to the mouse colon after intrarectal (lR) administration. The transgene was found highly expressed at days 1-3 and mainly confined to the colon. Gene expression was not only identified within the epithelium but also immediately beneath the epithelium, probably due to the penetration of Adv through epithelial cells. Mucosal immune responses were examined in an antigen model using ovalbumin (OV A). After IR immunization with Adv encoding OVA (AdOV A), the frequency of LP interferon (lFN)y secreting cells was detected as early as day 4, and continually progressed upward. The appearance of ILN IFN-y-secreting cells was transient, and this was mirrored by the ILN cytolytic activities. CDS+ T cells were stimulated to produce IFN-y, and cytolytic activities depended largely on CDS. Also, IR immunization with Adv induced Thl T-cell responses and local production of specific IgA. When challenged with recombinant vaccinia virus expressing OV A, immunized mice completely controlled local viral infection and prevented virus dissemination from the rectal tissue. Thus, an infectious mouse model of herpes simplex virus type 2 (HSV -2) given via the rectum was developed in the study and used to further validate the IR immunization regimen. Mice immunized with Adv encoding glycoprotein B (AdgB) were protected from rectal challenge of HSV -2 at absolute lethal doses. Clinical pathology and virus replication were remarkably reduced and virus-shedding period was significantly lessened. CDS+ T cells, IFN-y and interleukin (lL)-12 appeared to play an essential role in such protective immunity. Protection of mice against HSV-2 challenge in the vaginal tract was also achieved, thus indicating that rectal immunization could also confer protective genital immunity. In comparison with the intranasal (distant mucosal) and subcutaneous (systemic non-mucosal) immunization, rectal immunization proved to be more effective in the induction of rectal immune responses including the frequency of IFN-y secreting cells and IgA production, and to provide better protection against rectal or vaginal HSV-2 challenge. All these results underscored the importance of applying local mucosal immunization to induce mucosal immunity at both rectal and genital tracts. In conclusion, IR administration with Adv by the new delivery method efficiently transferred antigen genes into the rectal mucosa and elicited protective local immunity to virus challenge. The present study provided evidence that rectal (local mucosal) immunization regimen was a better vaccination strategy than distant mucosal and systemic non-mucosal immunization to provide both rectal and genital protection against viral infection. Thus, the present approach supports the view that route is a critical determinant of vaccination and, furthermore, represents fertile ground for future studies of mucosal vaccination via the rectal mucosa.</p> / Doctor of Philosophy (PhD)

<p>Asthma and other allied allergic diseases represent a significant burden to health care and are recognized as endemic in the Western World. While a diverse array of effective pharmacopoeia provides reprieve from symptoms, no preventive or curative therapies are currently available. This is in part due to the paucity of understanding how allergic diseases develop. Recently, remarkable progress has been made in this regard, largely due to the discovery of regulatory mechanisms that control responsiveness to antigen in the airway. Indeed, to understand fully why people develop asthma requires an understanding of both allergic sensitization and tolerance. Work presented in this thesis contributes to our knowledge of inhalation tolerance. Using a classical mouse model of allergic airways inflammation, we show in Chapter 2 that inhalation tolerance is a persistent and active process as it prevents the generation of airway eosinophilia, antigenspecific IgE and airway hyperresponsiveness upon secondary immunogenic challenge, independently of IL-lO or IFN-y. Building on these observations, in Chapter 3 we show in a mucosal model of allergic sensitization that inhalation tolerance cannot be broken with the expression of GM-CSF, a potent growth factor and cytokine that has been associated with asthma and allergy in both human and animal subjects. However, concomitant expression of decorin, a natural inhibitor of TGF-f3, reverses inhalation tolerance, thus implicating TGF-f3 as putatively important in regulating responsiveness in the airway. In Chapter 4, we identify an alternative mode of tolerance. We show that chronic exposure to innocuous antigen in sensitized mice does not lead to chronic inflammation but to abrogated eosinophilia that can, nevertheless, be reversed with the expression of GM-CSF. Collectively, these findings enrich our understanding of tolerance and provide a framework for new discoveries that may, ultimately, lead to novel and powerful therapies for allergic disease.</p> / Doctor of Philosophy (PhD)

<p>Cancer cell metastasis to bone is a frequent occurrence with many common-human malignancies, including carcinomas of the prostate and breast. Bone metastasis may be promoted by the phenotypic responses of cancer cells to factors which are present within the bone microenvironment. This thesis describes in vivo and in vitro efforts to test the hypothesis that bone matrix proteins promote the growth and adhesiveness of metastatic cells. An animal model was developed to examine the growth properties of rat mammary cancer cells (Walker 256, W256) in metastatic target organs (Chapter 1). Rats were injected intramuscularly with W256 celIs, which formed spontaneous metastases in the skeleton, liver, lungs, and kidneys within 7-10 days. Skeletal metastasis was associated with a progressive loss of trabecular bone. Within the skeleton, W256 cells which were immediately adjacent to trabecular bone had a 30% greater growth rate than did W256 cells located >50 μm from bone surfaces (p<0.05). These data established the first animal model of spontaneous bone metastasis, and provided preliminary evidence that bone matrix is a possible mitogen for metastatic cancer cells in vivo.</p> <p>The mitogenic activity of bone matrix may be enhanced by osteoclastic bone resorption (1). To test this hypothesis, bone resorption was stimulated by injecting rats with Leydig tumor cells (Chapter 2). Leydig tumor burden was associated with decreased trabecular bone volume, fewer osteoblasts, and more osteoclasts. Leydig tumor burden was associated with a 56% increase in the growth rate of W256 cells in the skeleton (p<0.05), while W256 cell growth rate in the liver, lungs, and kidneys were not affected. The selective growth promotion of W256 cells in bone suggests the existence of a mitogen for W256 cells which is released from bone matrix during resorption.</p> <p>Previous in vitro data suggest that this putative bone-derived mitogen may be transforming growth factor β (TGF-β) (1), a factor which can stimulate oncogene expression. Media conditioned by resorbing fetal rat calvarial bones induced a rapid increase in W256 cell expression of c-myc oncogene mRNA and of nuclear c-myc protein (Chapter 3). Purified TGF-β also stimulated c-myc mRNA expression, suggesting that the mitogenic stimulation of W256 cells by bone-derived conditioned media may involve a signaling pathway which is also utilized during TGF-β-induced growth stimulation.</p> <p>Bone resorption may be a sufficient but unnecessary growth stimulus for metastatic cells in vivo. To test this hypothesis, bone resorption in rats was inhibited in vivo with the bisphosphonate APD prior to their inoculation with W256 cells (Chapter 4). APD blocked the pathologic bone resorption normally associated with W256 tumor burden. However, the skeletal tumor burden in APD-treated rats was 2.6-fold greater than in untreated rats. Tumor burden in other organs was unaffected by APD. W256 cells in the skeletons of APD-treated rats had a 55% greater growth rate than did W256 cells in the skeletons of controls. W256 cells located adjacent to trabecular bone had greater growth rates than did W256 cells>50 μm from bone, irrespective of APD treatment (p<0.05). These data suggest that bone matrix can exert a mitogenic influence on cancer cells without undergoing resorption.</p> <p>Bone-derived factors are also capable of promoting adhesion (2), and the adhesion of metastatic cells to bone matrix could promote their skeletal localization. An in vitro model of bone matrix was developed to identify potential ligands which could support the adhesion of metastatic cells (Chapters 5 and 6). The extracellular matrix deposited by human U2OS osteosarcoma cells supported the rapid adhesion of human PC-3 prostatic carcinoma cells. Antibodies directed against the integrin-type collagen receptor α2β1 inhibited PC-3 cell adhesion to U2OS matrix and to purified type I collagen, as did a collagen-derived peptide sequence (DGEA) which inhibits α2β1 function. TGF-β treatment caused a selective increase in α2β1 integrin expression, which was effected by increased rates of de novo synthesis of both α2 and β1 integrin subunits. The induction of α2β1 was associated with similar increases in the adhesion of PC-3 cells to U2OS matrix and to purified type I collagen. These data suggest that type I collagen may represent an important bone-derived adhesive ligand for metastatic cells. The abundant expression of type I collagen and of TGF-β in bone may promote bone metastasis by promoting the adhesion of metastatic cancer cells to bone matrix.</p> / Doctor of Philosophy (PhD)

<p>The general focus of my project was the production of recombinant adenovirus vectors for use in inmmunotherapy of cancer. The basic strategy involved the infection of tumour cells with Ad-vectors expressing cytokines, inducing a local anti-tumour response. The cytokine of interest for my project was tumour necrosis factor alpha (TNFα), which I used for treatment of a murine transgenic model of breast cancer. TNFα is a pluripotent cytokine with a wide variety of physiological functions including antitumour activity. TNFα was originally discovered through the anticancer activity of sera of mice treated with endotoxin (Carswell et al., 1975). There are two known cell surface receptors for TNFα termed p55 and p75. Both receptors signal a variety of functions and some redundancy exists between them. However the p55 TNF receptor is the major activator of cytotoxicity and cytokine secretion, while the p75 TNF receptor is primarily responsible for proinflammatory and lymphoproliferative signals. Perhaps the major limiting factor affecting the use of TNFα in tumour therapy is its systemic toxicity, through the induction of septic shock and cachexia (Tracey, 1995; Tracey et al., 1986). We have been investigating techniques which would reduce the systemic side effects of TNFα, while retaining its antitumour activity. We found that local expression of TNFα from within a tumour (transduced cells) alone is not enough to eliminate its lethal side effects, therefore two other approaches are being investigated in an attempt to deal with systemic toxicity induced by TNFα. The first was the construction of an Ad vector expressing a membrane bound mutant of murine TNFα (see chapter III). The second approach involved specific targeting of the two cell surface receptors of TNFα. To accomplish this, we used Ad vectors expressing human TNFα, and a novel p75 TNF receptor specific mutant of murine TNFα for use in tumour immunotherapy (See chapters IV and V). It was found that restricting TNFα to the membrane produced a marked reduction in lethality while retaining near normal antitumour activity. Targeting the p55 TNF receptor proved to be ineffective, while targeting the p75 TNF receptor drastically reduced the lethality of the cytokine while retaining some antitumour activity. However the overall efficacy of this therapeutic technique was poor, as only a low percentage of mice were cured with our Ad-TNF vectors.</p> / Doctor of Philosophy (PhD)

<p>The studies described in this thesis were undertaken to examine the mechanism of lactate production in exercise from a standpoint of enzymatic control and investigate the two rate-limiting enzymes which regulate glycogen breakdown and oxidation, glycogen phosphorylase and pyruvate dehydrogenase (PDH). The purpose was to establish the theory that it is the differential rate of flux through these enzymes which determines the extent of lactate accumulation in situations where significant lactate production occurs such as during 30-s bouts of maximal intermittent exercise and during exercise in hypoxia. In the first study, at the onset of maximal exercise, the transformation of PDH was delayed relative to phosphorylase accounting for a greater lactate accumulation. PDH was maximally transformed after 15s and remained elevated until 30s, whereas the transformation of phosphorylase remained elevated at 15s and reverted to resting levels after 30s. Before the third bout of exercise, PDH was nearly 50% transformed and was fully transformed after only 6s, whereas phosphorylase remained at resting levels. The regulation of these enzymes at the last 15s of the first bout and throughout the third bout is consistent with a reduction in glycogenolysis and increased pyruvate oxidation leading to less lactate accumulation. In the second study, at the onset of exercise in hypoxia, there was a delayed transformation of PDH relative to normoxia, accounting for the greater lactate accumulation typically observed in hypoxia. The results from these first two studies suggested that the metabolic inertia of PDH activation may be a factor in the mechanism of lactate production in both maximal exercise and in hypoxia. The third study sought to remove the metabolic inertia of PDH activation during exercise in hypoxia with the infusion of dichloroacetate (DCA). DCA fully transformed PDH at rest and at the onset of exercise in hypoxia. By removing the metabolic inertia of PDH activation with DCA or with prior exercise as in the third bout of maximal exercise in the first study, lactate production was significantly reduced. The findings from these studies demonstrate that the mechanism of lactate production is the result of a mismatch between the rates of pyruvate production and pyruvate oxidation due to the differential catalytic rates between glycogen phosphorylase and PDH under conditions where oxygen is presumed to be limiting such as maximal exercise and exercise in hypoxia. These results contradict the theory that an O2 limitation is the main cause for lactate production ("anaerobic metabolism") and suggest that the metabolic inertia of enzyme activation also plays a role. The data are indicative of a central role for PDH and phosphorylase in the mechanism of lactate production and further question whether O2 is limiting during exercise in healthy humans.</p> / Doctor of Philosophy (PhD)

<p>In our studies, we have used adenoviruses containing the genes for Angiostatin (Ad-Angiostatin) and IL-12 (Ad-IL12) for tumor therapy. The biological activity of the Ad-Angiostatin has been characterized using an artificial matrix which promotes the growth of blood vessels (Matrigel). We observed suppression of vessel growth in the Matrigel following treatment with Ad-Angiostatin demonstrating the inibitory activity of this virus. Treatment of growing tumor in mice with either Ad-Angiostatin or Ad-IL12 resulted in modest delays in tumor growth compared to tumors treated with viruses which did not contain any genes. Ad-IL-12 treatment also causes tumors to regress in a small fraction of the treated animals (13%). However, when used in combination, treatment with Ad-Angiostatin and Ad-IL12 resulted in tumor regression in 54% of the cases, a strong anti-tumor response by killer white blood cells, and the cured animals were resistant to further outgrowth of tumor. These results are the first to demonstrate the usefulness of combining angiogenesis inhibitors with cytokines using gene therapy. The rationale for this therapy is to limit the tumor size by attacking the vasculature with angiostatin, allowing the Interleukin-12 to activate killer white blood cells to respond against foreign products present in the tumor tissue. In this manner, the potential for the white blood cells to reject the tumor is increased as there is less tumor mass present. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>Lipopolysaccharide (LPS), a component of the outer membrane of gramnegative bacteria, is a potent inflammatory stimulus responsible for a number of clinically critical airway conditions including gram-negative pneumonia, gram-negative septic lung injury and septic adult respiratory distress syndrome (ARDS). These conditions are all characterized by activation of alveolar monocytes/macrophages and massive infiltration of polymorphonuclear leukocytes (PMNs). While it is generally agreed that alveolar macrophages have a central role in the initiation of neutrophil accumulation and that the contents released from PMNs play a major part in causing tissue injury, the molecular mechanisms underlying these processes remain incompletely understood. Recently, some cytokines (polypeptide hormones) such as interleukin-1 (IL-1), tumor necrosis factor α (TNFα), interleukin-8 (IL-8) and interleukin-6 (IL-6), have been implicated in these processes, yet the expression of these cytokines by airway cells in response to LPS still remains to be fully elucidated. Thus, both in vitro and in vivo studies were undertaken to investigate the effect of LPS on cytokine gene expression and protein production by airway cells and tissues.</p> <p>To investigate the regulation of cytokine expression in alveolar macrophages by LPS and extracellular matrix (ECM) components, LPS stimulated rat alveolar macrophages were maintained on different substrates: plastic, collagen and airways fibroblast-derived ECM (fECM). It was found that adherence to plastic induced IL-1β expression and that LPS further enhanced this expression in a time-dependent manner. In contrast, significant expression of IL-6 mRNA was observed only in LPS-stimulated alveolar macrophages. Adherence to collagen or fECM evoked a stronger IL-1β mRNA expression as compared to adherence to plastic. Only cells cultured on fEeM, however, retained maximal responsiveness to LPS stimulation over a period of five days in culture.</p> <p>To determine whether LPS can directly act on airway-derived fibroblasts, nasal, bronchial and lung fibroblasts were exposed to LPS and expression of several cytokines including granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-8 and IL-6 was assessed. While fibroblasts of all three anatomic sites produced significantly increased amounts of cytokines in response to IL-1, only upper airway-derived nasal fibroblasts could synthesize significant quantities of cytokines upon LPS stimulation.</p> <p>Furthermore, the interaction of airways structural epithelial cells and fibroblasts with monocytes/macrophages was examined. Human peripheral blood monocytes were cultured with either nasal epithelial cell- or fibroblast-derived conditioned medium, and survival, proliferation and differentiation of monocytes then analyzed. Both macrophage colony-stimulating factor (M-CSF) and particularly, GM-CSF released by airway structural cells were found to dramatically promote survival and differentiation, but not proliferation, of monocytes.</p> <p>To investigate the temporal sequence of cytokine induction upon LPS exposure in vivo, a rat model of acute lung inflammation was established. Kinetics and cellular origin of cytokines were examined. LPS markedly evoked an early expression of TNFα and macrophage inflammatory protein-2 (MIP-2; the rodent functional equivalent of human IL-8), followed by IL-1β and IL-6 but not RANTES (a T cell/monocyte chemotactic cytokine) or transforming growth factor β₁ (TGFB₁) expression. Alveolar macrophages represented the most significant source of cytokines shortly after LPS challenge. At later times, the infiltrating PMNs were the most significant source of cytokines in the lung.</p> <p>In conclusion, these data suggest that (a) the alveolar macrophage can respond directly to LPS stimulation by elaborating and releasing cytokines; (b) this cytokine response can be modulated by the extracellular environment in which alveolar macrophages reside; (c) only the fibroblast population in the upper airway, in close proximity to the external environment, is capable of responding to LPS exposure by cytokine release. This particular fibroblast population may be thus directly involved in the initiation of acute airways inflammation; (d) monocytes/macrophages and airway structural cells communicate with each other. This interaction is accomplished in part through cytokines released from the structural cells; and (e) LPS evokes a sequential and specific cytokine response in the airways in vivo. TNFa and MIP-2 appear to play a major early role in eliciting the neutrophilic response. Consistent with in vitro findings, alveolar macrophages serve as a significant source of cytokines in vivo prior to recruitment of PMNs. Thereafter, PMNs serve as the other significant source of cytokines in vivo, suggesting that these cells likely contribute to the amplification of the response to LPS in an autocrine and paracrine fashion.</p> / Doctor of Philosophy (PhD)

<p>Herpes simplex virus type 1 and adenovirus type 5 are nuclear DNA viruses that encode a number of regulatory polypeptides that serve to facilitate expression of the viral genome at the expense of host gene expression. These viruses depend on cellular transcriptional apparatus, and viral regulatory proteins flmction primarily to ensure that host factors engage the viral template and that viral genes are expressed in the correct spatial and temporal sequence. The results presented in this thesis demonstrate that regulatory proteins encoded by herpes simplex virus and adenovirus modulate the expression of two classes of cellular genes: the globin genes and Alu repetitive sequences. Herpes simplex virus gene products were required to stimulate the expression of human α-globin, rabbit β-globin genes and Alu elements introduced into cells as part of the herpes genome. In addition, infection with herpes simplex virus or adenovirus stimulated expression of host α-globin and Alu sequences and de novo synthesized viral gene products were required for induction of these cellular genes. Viral induction of α-globin and Alu expression are both unusual instances of activated gene expression: viral infection allows the α-globin gene to escape its erythroid~restricted transcription pattern, and the viral activation of RNA polymerase III transcription of Alu sequences is the first instance of high level class III transcription of these repetitive DNA elements in vivo. The identification of the herpes implex virus and adenovirus gene products which mediate the transcriptional activation of these host sequences has contributed to the understanding of the mechanisms which regulate expression of α-globin genes and Alu repetitive elements and also of the mechanisms by which viral regulatory proteins modulate gene expression.</p> / Doctor of Philosophy (PhD)