Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe complication of dental treatment in 1-10% of patients previously treated with intravenous bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates have the potential to depress the immune system in mice and a subset of patients, possibly contributing to the pathogenesis of BPONJ.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.

The aim of this project was to study the mechanisms of disease resistance in plaice (<i>Pleuronectes platessa</i> L.). Within this broad area, the work concentrated on investigating the migration, phagocytic activity and luminol-dependent chemiluminescence (CL) of plaice phagocytic leucocytes <i>in vitro</i>. In these studies two sources of leucocytes were used: (i) glycogen-elicited, peritoneal exudate cells and (ii) kidney leucocytes that had been purified on a discontinuous Percoll gradient. Both cell isolates comprised of at least 70% neutrophils. Several factors were found to chemoattractants for both peritoneal and kidney neutrophils. Endogenous attractants included leukotriene B<SUB>4</SUB> and two neutrophil-derived factors, which differed in their solubilities in organic solvents. Exogenous attractants included the formyl-peptide, N-formylmethionyl-leucyl-phenylalanine, and an organic solvent soluble factor released by viable <i>Vibrio alginolyticus</i>. The interactions of these attractants and their <i>in vivo</i> activities were also investigated. The migration of neutrophils <i>in vitro</i> was significantly reduced in the presence of cortisol at levels equivalent to those found in stressed plaice (> 400 ng/ml). The opsonic activity of plaice serum was investigated by analysing the phagocytosis and CL of plaice neutrophils. Sheep erthrocytes were only opsonized by the presence of specific antibody and an activatable factor(s) found in normal serum. The bacterium, <i>V. alginolyticus</i>, was opsonized by a factor(s) in normal serum, the activity of which was not further enhanced by the presence of specific antibody. In fact, two different opsonins were identified in normal plaice serum: (i) a very heat-stable activity (stable at 70<SUP>o</SUP>C for 30 min) recognised by kidney neutrophils and (ii) a heat-labile activity (50<SUP>o</SUP>C for 30 min) recognised by peritoneal neutrophils. Plaice neutrophils were shown to produce several reactive oxygen species: superoxide anion (O<SUP>-<SUB>2</SUB></SUP>), hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>) and their derivatives, possibly including hydroxyl radical (OH<SUP>bullet</SUP>).

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "December 1996." Typescript. Vita. Includes bibliographical references (leaves 118-129). Also available on the Internet.

No description available.

Vascular hyperpermeability is a common characteristic among many tumour types, especially those that grow in ascites form. With these, the exudate that flows out of the circulation collects as ascites fluid in the cavities within which these tumours are growing. In the past, this hyperpermeability has been attributed to the production of vascular permeability factor (VPF) by tumours. VPF has been found to bind to endothelial cells and lead to an increased vascular permeability. In the present study, the role of polymorphonuclear leukocytes (neutrophils) in tumour vascular hyperpermeability was investigated. Hey-3 tumour cells were grown into masses on the chick embryo chorioallantoic membrane (CAM). Interstitial neutrophilia was found to be a common feature at the tumour-host interface. Horseradish peroxidase was injected into the circulation and allowed to perfuse for five minutes. The density of labelled vesicles within the endothelial cell cytoplasm was calculated to be 0.99 +/-0.28 vesicles/μm². This vesicular density was comparable to that of N-formyl-methionine-leucine-phenylalanine (a chemotactic peptide for neutrophils)-treated CAM (1.04 +/-0.09 vesicles/μm²), but very different from control CAM (0.51 +/- 0.09 vesicles/μm²). In order to rule out any immune response to foreign cells, immune hepatocyte masses were grown on the CAM and vesicular density was calculated to be 0.54 +/-0.03 vesicles/μm². Through chemotaxis assays with the Boyden chamber, it was observed that Hey-3 tumour cells in culture were producing a chemotactic factor that is an attractant for human neutrophils. Once in the area, neutrophils do possess the potential to increase vascular permeability. Thus, neutrophils play a role in vascular endothelial hyperpermeability in tumours. / Thesis / Master of Science (MS)

Neutrophils are known to be key innate defenders through performing vital and diverse functions such as degranulation, oxidative burst, and generation of extracellular trap (NET). Recent data suggest that neutrophils may also play key roles in modulating tissue inflammatory/immune environment by secreting soluble mediators as well as surface-attached co-activators. Furthermore, neutrophils may adopt distinct functional states either conducive or detrimental for tumor-growth through cellular contact with cancer cells and/or other immune cells such as T helper cells. However, molecular mechanisms that modulate functional adaptations of neutrophils are not well understood. The objective of my thesis is to identify the role of Tollip, a novel TLR signaling adaptor molecule, in modulating neutrophil functions by suppressing the inflammatory signaling pathway. Preliminary data from our lab suggest Tollip deficient neutrophils may be programed to exhibit enhanced anti-tumor activities. Based on these novel findings, I tested the hypothesis that neutrophils also have subsets with different functions similar to monocyte/macrophages, and Tollip deficient neutrophils may be programmed into an enhanced anti-tumor state through upregulating inflammatory signaling processes and mediators. / Ph. D. / Cancer immunotherapy gained instant popularity overnight after former president of the United States, Jimmy Carter, announced successful treatment of his metastatic melanoma. Since then, FDA has approved the first immunotherapy in the summer of 2017. Current cancer immunotherapy focuses heavily on the potential of T cells to target unregulated cells in the host. However, time and specificity have proven a difficult challenge to overcome. Innate immune cells may circumvent these challenges and present equal potential as therapy in the fight against cancer. Neutrophils are one of the innate immune cells that provide first line of defense against pathogens. Neutrophils can clear danger or maintain the situation until more cells arrive to help clear the danger. They were originally thought to confer simple and transient effects, but emerging data suggest they may have more diverse role in host defense. We propose programming neutrophils will make the cells less susceptible to cancer manipulation and provide enhanced protection against cancer establishment and maintenance.

Neutrophil extracellular traps (NETs), chromatin released by activated neutrophils, were first described for their antimicrobial properties. NETs have a backbone of DNA and histones lined with microbicidal proteins such as neutrophil elastase. NET release has pathological consequences, particularly within blood vessels where NETs can trap red blood cells and platelets, thus contributing to thrombosis (Chapter 1-Overview). NET formation (NETosis) is an active and coordinated biological process involving many enzymatic components. One enzyme in particular, peptidylarginine deiminase 4 (PAD4), citrullinates histones and is required for chromatin decondensation during NETosis. Neutrophils from PAD4-deficient mice are unable to form NETs. We obtained these mice from our collaborator Dr. Yanming Wang, and thus were able to compare PAD4-/- mice to wild-type (WT) mice in mouse models where NETs are formed. These studies have allowed for investigation of the biological relevance of PAD4 and NETs in vivo in thrombotic and/or inflammatory disease. This dissertation focuses on mouse models of deep vein thrombosis and of sepsis. In venous stenosis, thrombosis is initiated by restricting blood flow in the inferior vena cava (IVC). Here, PAD4-/- mice were greatly protected from thrombus formation (Chapter 2). Leukocyte rolling and platelet plug formation in response to vessel injury were unaffected, indicating that endothelial and platelet activation occurred normally in these mice. The mice did not exhibit any defects in hemostasis, and could be induced to produce deep vein thrombi by infusion of WT neutrophils that formed NETs as a part of the thrombus scaffold. Because there is potential to develop anti-NET therapies in thrombosis, I investigated if NET-deficiency would render mice immunocompromised (Chapter 3). PAD4-/- mice had similar mortality in the cecal ligation puncture model, and they were protected from shock in an LPS sepsis model where NETs are released in the absence of live bacteria. Therapies aimed at NET prevention or destruction would likely be beneficial without compromising host immunity. Thus, in summary, studying PAD4-deficient mice has revealed the impact of NETs in thrombotic/inflammatory disease and identified PAD4 as an attractive therapeutic target.

Obesity is increasing worldwide, and the associated health-risks are also on the rise. Eventually, obesity related tissue damage leads to complications such as chronic inflammation, diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Adipose tissue expansion in obesity triggers specific mechanisms that cause tissue damage. The immune system is especially agitated with excessive fat accumulation, which triggers inflammation and subsequent immune cell infiltration of tissue. Neutrophils are a major immune cell that cause damage in obesity, and the protease neutrophil elastase (NE) is a major neutrophil released factor of tissue damage. The goal of this study is to use tissue extracted from neutrophil elastase knockout (NEKO) mice that have been fed a high-fat high-fructose diet (HFHFD), and compare them to wild-type (WT) mice fed a normal chow diet (NCD), high-fat diet (HFD), and HFHFD to understand the effect of neutrophil elastase damage in obesity. Tissue from aged (NEKO) mice will also be examined to evaluate the role of neutrophils and NE in tissue damage in aging and obesity. Mice in these experimental groups were sacrificed and had their tissue extracted for various staining protocols to discover the extent of tissue damage and immune cell infiltration between mice with and mice without NE. One experiment had 4 different diets fed to mice. The other experiment had mice aged for 2 years, and mice aged for 3 months and 4 months. Mice from the first experiment were fed for 4 months and separated into 4 groups based on diet, WT-NCD, WT-HFD, WT-HFHFD, and NEKO-HFHFD. Our data indicates that, in comparison with WT-HFHF mice, NEKO-HFHFD mice had less steatosis, fibrosis, immune cell infiltration, and apoptosis within the liver. Neutrophil infiltration into the liver is increased by the HFHFD diet. HFHFD diet also stimulates fibrosis, as indicated by collagen deposition in the liver. Neutrophil accumulation is also associated with the increase of macrophages and CD4 Th Cells in the liver, particularly in WT mice fed the HFHFD. Interestingly, the liver from NEKO-HFHFD mice had dramatically reduced infiltration of neutrophils, macrophages, and CD4+ Th cells. Our data suggests that NE is required for HFHFD induced inflammation and fibrosis in the liver. Mice from the second experiment were split into 3 groups based on age, WT-Young (3 months and 4 months), WT-Old, and NEKO-Old. All groups were fed the same normal chow diet, but WT Old and NE KO Old were both aged to 2 years old. Our data revealed that NE deletion in aged mice reduced fibrosis, elastin fragmentation, calcification, and presence of NE within the aorta. While part of the mechanism for neutrophil elastase related tissue damage has been explored through this one-year master degree research project, more work is needed to fully understand how NE is stimulated and causes tissue damage. Future work should examine the potential interaction between neutrophils and other immune cells in obesity and aging. / 2022-06-04T00:00:00Z