Apoptosis is a physiological form of cell death. It is a highly evolutionarily conserved process that is non-inflammatory or anti-inflammatory in nature. This anti-inflammatory nature of apoptosis is evident by the fact that neutrophils are histologically absent from sites where homeostatic apoptosis rates are high. The rapid phagocytosis of apoptotic cells as a means to prevent the release of noxious inflammatory compounds also accounts for the anti-inflammatory environment of such sites. However, the mechanisms that enable mononuclear phagocytes to migrate to sites where homeostatic apoptosis rates are high, and not granulocytes, the professional phagocytes that accumulate at sites of inflammation, have not been determined yet. Using Burkitt’s lymphoma (BL) as a model of apoptosis, the aim of this thesis was to identify the regulatory mechanisms or factors underlying the non-phlogistic features of sites where homeostatic apoptosis rates are high and in particular, those preventing the recruitment of neutrophils - a major granulocyte subclass to these sites. BL is a highly aggressive B cell lymphoma that is mainly characterised by a high rate of apoptosis. By carrying out a series of in vitro chemotaxis assays and biochemical approaches, it was found in this thesis that BL cells actively inhibit neutrophil migration by releasing factors that were identified to be lactoferrin, a 80 kDa iron-binding glycoprotein with anti-bacterial and anti-inflammatory properties. It was further demonstrated that lactoferrin selectively inhibited migration of granulocytes (both neutrophils and eosinophils) but not mononuclear phagocytes and this effect was irrespective of its iron saturation status and the chemoattractant used. Also, lactoferrin potently inhibited neutrophil migration, as assessed by thioglycollate-induced in vivo model of mouse peritonitis. This anti-inflammatory function of lactoferrin was attributed to its effect on granulocyte signalling pathways that regulate cell adhesion and motility. Finally, it was demonstrated that in cell types of diverse lineages, induction of apoptosis results in de novo synthesis and secretion of lactoferrin. In subsequent proliferation assays determining the in vitro growth of a number of BL cell types, it was demonstrated that lactoferrin is an essential component of BL cells and promotes their proliferation, as its antibody-mediated neutralisation or shRNA-mediated expression knockdown, reduced BL cell growth. Together, the results of this thesis identified lactoferrin as one of the few characterised antiinflammatory components of the apoptosis milieu that negatively regulate granulocyte migration. This effect may provide opportunities for broad therapeutic interventions concerning the use of lactoferrin in chronic inflammatory conditions characterised by aberrant neutrophil influx as well as atopic allergic disorders, such as asthma. Moreover, based on the tumour-supporting role of lactoferrin described in this study, targeting its expression in tumours could lead to tumour regression and thus, be a promising therapeutic molecule in tumour immunotherapy.

This thesis embodies research investigating the role that bacterial biofilms play in the pathogenesis of chronic rhinosinusitis (CRS). It focuses on their detection on the sinus mucosa of CRS patients and the implications of their presence. Finally, it addresses deficiencies in the innate immune system that may predispose to their development in this condition. Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self-produced matrix and irreversibly attach to a surface. Their extreme resistance to both the immune system as well as medical therapies has implicated them as playing a potential role in the pathogenesis of many chronic diseases. Although their role in many diseases is now well established, their objective presence and importance in CRS remains largely unknown. Chapter 1 of this thesis reviews the current literature pertaining to CRS and biofilms and critically evaluates the small body of research relating to this topic. Chapter 2 describes the development of a sheep model to study the role of bacterial biofilms in rhinosinusitis. It compares the use of traditional electron microscopy (EM) and more recent confocal scanning laser microscopy (CSLM) in the detection of biofilms on the surface of sinus mucosa. The results of this study inferred a causal relationship between biofilms and the macroscopic changes that accompany rhinosinusitis. Furthermore it illustrated the superiority that CSLM has over EM in the imaging of biofilms on sinus mucosa Chapter 3 and 4 outline the results of human studies utilizing the more objective CSLM to evaluate the prevalence of bacterial biofilms on the sinus mucosa of CRS patients and their effect on post-operative mucosal healing. The results of these studies demonstrated a biofilm prevalence of approximately 50% in the CRS population studied and suggested, that biofilm presence may predispose to adverse post-operative outcomes following sinus surgery. Chapter 5 and 6 describe experiments examining the level of the innate immune system’s anti-biofilm peptide lactoferrin, in patients with CRS. Lactoferrin was found to be downregulated at both an mRNA and protein level in the majority of CRS patients, with biofilm positive patients demonstrating the most significant reduction. In summary, this thesis provides further evidence that bacterial biofilms play a major role in the pathogenesis and disease persistence in a subset of CRS patients. Deficiencies in components of the innate immune system, such as lactoferrin, may play an important role in the predisposition of certain individuals to the initial development of bacterial biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346621 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine 2008

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Food Science." Includes bibliographical references.

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.

This thesis embodies research investigating the role that bacterial biofilms play in the pathogenesis of chronic rhinosinusitis (CRS). It focuses on their detection on the sinus mucosa of CRS patients and the implications of their presence. Finally, it addresses deficiencies in the innate immune system that may predispose to their development in this condition. Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self-produced matrix and irreversibly attach to a surface. Their extreme resistance to both the immune system as well as medical therapies has implicated them as playing a potential role in the pathogenesis of many chronic diseases. Although their role in many diseases is now well established, their objective presence and importance in CRS remains largely unknown. Chapter 1 of this thesis reviews the current literature pertaining to CRS and biofilms and critically evaluates the small body of research relating to this topic. Chapter 2 describes the development of a sheep model to study the role of bacterial biofilms in rhinosinusitis. It compares the use of traditional electron microscopy (EM) and more recent confocal scanning laser microscopy (CSLM) in the detection of biofilms on the surface of sinus mucosa. The results of this study inferred a causal relationship between biofilms and the macroscopic changes that accompany rhinosinusitis. Furthermore it illustrated the superiority that CSLM has over EM in the imaging of biofilms on sinus mucosa Chapter 3 and 4 outline the results of human studies utilizing the more objective CSLM to evaluate the prevalence of bacterial biofilms on the sinus mucosa of CRS patients and their effect on post-operative mucosal healing. The results of these studies demonstrated a biofilm prevalence of approximately 50% in the CRS population studied and suggested, that biofilm presence may predispose to adverse post-operative outcomes following sinus surgery. Chapter 5 and 6 describe experiments examining the level of the innate immune system’s anti-biofilm peptide lactoferrin, in patients with CRS. Lactoferrin was found to be downregulated at both an mRNA and protein level in the majority of CRS patients, with biofilm positive patients demonstrating the most significant reduction. In summary, this thesis provides further evidence that bacterial biofilms play a major role in the pathogenesis and disease persistence in a subset of CRS patients. Deficiencies in components of the innate immune system, such as lactoferrin, may play an important role in the predisposition of certain individuals to the initial development of bacterial biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346621 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine 2008

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.

Universiẗat, Diss., 2003--München.

Allergic contact dermatitis is an important occupational health disease. It represents a useful experimental paradigm in which the mechanisms and characteristics of cutaneous immune responses can be investigated. This thesis has focused on the sensitization phase of contact allergy, including Langerhans’ cell (LC) migration, cytokine expression and the ability of the protein lactoferrin (LF) to modulate aspects of these processes. Lactoferrin was originally identified as an antimicrobial protein. However, it is being recognized increasingly to have immunomodulatory effects on the cells of the immune system. Migration of LC in mice and in humans is mediated via two independent cytokine signals delivered by tumour necrosis factor (TNF)-α and interleukin (IL)-1β, which were thought to derive from keratinocytes and LC, respectively. Further, topical application of LF was shown to inhibit LC migration in both man and mouse potentially through the inhibition of de novo TNF-α production. The inhibitory effect of LF on LC mobilization induced by the contact allergen 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) has been confirmed in these investigations. Conversely, LF did not inhibit LC migration triggered by another contact allergen, 2,4-dinitrochlorobenzene (DNCB). That result prompted a comparison between oxazolone and DNCB with respect to their ability to induce LC migration and to provoke cutaneous cytokine production. It was discovered that DNCB induced LC mobilization in the absence of TNF-α signalling. Moreover, exposure to superoptimal doses of oxazolone resulted in TNF-α independent LC migration. Further experiments revealed that TNF-α independence might be mediated partially by the elevated concentration of IL-1β produced in the skin following exposure to DNCB and these superoptimal concentrations of oxazolone. Investigations of the immunomodulatory mechanism of LF in vitro demonstrated that it did not inhibit TNF-α production by THP-1 macrophages. On the contrary, LF was shown to stimulate TNF-α and IL-8 release by THP-1 macrophages in a dose dependent manner, via endotoxin-independent and nucleolin-dependent mechanism. Subsequently, the role of LF in modulation of keratinocyte activation was investigated. Keratinocytes expressed high levels of inducible TNF-α mRNA, however, this was not modulated specifically by LF. Additional examination of the effects of LF in vivo revealed that it inhibited cutaneous IL-17 and CXCL1 mRNA expression, induced by IL-1β and IL-1α, respectively. Lactoferrin treatment did not affect oxazolone-induced lymph node (LN) cell proliferation. However, it was demonstrated to decrease IL-17 production by LN cells 24h following exposure to oxazolone, which may be important in driving the vigour and/or quality of response to the contact allergen. Overall, these investigations have demonstrated a divergence within the family of contact allergens with regard to the requirement for TNF-α signalling for LC mobilization. It was established that when elevated concentrations of IL-1β are present LC migration can occur in the absence of TNF-α signalling. Moreover, a dual nature of LF, which can act in a stimulatory as well as inhibitory manner, was confirmed. These investigations have revealed a potential role for CXCL1 and IL-17 in the process of LC migration. Furthermore, it was shown that the inhibitory effect of LF on oxazolone induced LC migration might be mediated via its effect on IL-17.

BACKGROUND: Early identification of pediatric patients with Inflammatory Bowel Disease (IBD), including ulcerative colitis and Crohn disease, is important to help clinicians design optimal treatment regimens. Existing endoscopic techniques are effective in identifying disease activity. However, these methods are invasive, expensive, and less amenable to serial measurement. Recent studies have identified potential serologic and fecal biomarkers that may have the potential to provide clinicians with a more objective evaluation of disease activity. In the case of ulcerative colitis (UC), in which disease is confined to the large intestine, the information provided by fecal biomarkers is likely to be more specific than that provided by serologic biomarkers. Fecal lactoferrin (FLA) is one such biomarker that has shown to be useful not only in identifying levels of colonic inflammation, but also for use as a predictor of disease relapse and treatment efficacy. Measurement of fecal lactoferrin, in conjunction with information provided by other diagnostic modalities could expedite patient assessment and treatment. Additionally, it has been suggested that fecal lactoferrin levels may also provide prognostic information about response to treatment and disease outcome in pediatric patients with UC. The goal of this study is to explore the relationship between changes in FLA levels and response to medical therapy in hospitalized pediatric patients with UC. METHODS: Serial stool samples were collected daily from 10 patients admitted for management of severe active UC. Of these 10 patients, 3 responded favorably to standard treatment with intravenous corticosteroid therapy and were discharged to complete a course of oral steroids. 7 were unresponsive to steroid therapy and went on to require rescue (more intensive) medical therapy. Changes in FLA were correlated with steroid response and medical disposition at the time of discharge. RESULTS: A t-test was performed to determine the significance of the differences in percent change in FLA levels between patients discharged on steroids and patients discharged on rescue therapy. Patients discharged on steroids demonstrated a net decrease in FLA levels over the course of the first three days of steroid treatment while patients ultimately requiring rescue medical therapy demonstrated a net increase in FLA levels (mean values = -64.4% and +203.8%, respectively). A difference was found between the averages; however, this value did not reach statistical significance when analyzed with a t-test (p = 0.18). CONCLUSIONS: This study suggests that quantitative FLA levels may prove useful in predicting clinical course and discharge outcome in pediatric patients with ulcerative colitis. Future research in this field should seek larger sample sizes, increased longitudinal sample collection, and the potential for a composite assessment that will yield additional objective measures of disease activity.

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