Bovine respiratory disease complex is the most common disease that causes sig-nificant economic loss, typically in feedlot cattle. Current treatment methods are focused on reducing inflammatory responses, control of airway reactivity and improvement of pulmonary functions without potential side effects. Neutrophils are the key contributors in acute lung inflammation. However, activated neutrophils live longer and cause exces-sive tissue damage upon migration into lungs. Therefore, modulation of their migration and lifespan are attractive approaches in treatment strategies of bovine respiratory dis-ease. Nanotechnology holds significant potential to design new compounds by our ability to manipulate at the nanoscale. Helical rosette nanotubes are a class of novel, biologi-cally inspired, water soluble and metal-free nanotubes. I used helical rosette nanotubes conjugated to arginine-glycine-aspartic acid (RGD-RNT) to study their effects on neu-trophil chemotaxis, cell signaling and apoptosis. Bovine neutrophils exposed to 5% RGD-RNT reduced their migration in response to fMLP (formyl-Methionyl-Leucyl-Phenylalanine), compared to the non-treated group (P<0.001). This inhibitory effect was the same as that of groups treated with ERK1/2 inhibitor (UO126) and p38 MAPK in-hibitor (SB239063). In addition, the phosphorylated ERK1/2 and p38 MAPK for the first time were quantified by sandwich ELISA to elucidate the mechanism of neutrophil mi-gration. The phosphorylation of both the ERK1/2 and p38 was inhibited at 5 minutes by RGD-rosette nanotubes (P<0.05). Furthermore, integrin ÑvÒ3 is possibly involved in mi-gration of bovine neutrophils. Moreover, RGD-RNT did not induce apoptosis of bovine neutrophils which was inversed by pre-exposing them to LPS for 30 minutes (P<0.001). These experiments provide the first evidence that RGD-rosette nanotubes suppress phos-phorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils.

Bovine respiratory disease complex is the most common disease that causes sig-nificant economic loss, typically in feedlot cattle. Current treatment methods are focused on reducing inflammatory responses, control of airway reactivity and improvement of pulmonary functions without potential side effects. Neutrophils are the key contributors in acute lung inflammation. However, activated neutrophils live longer and cause exces-sive tissue damage upon migration into lungs. Therefore, modulation of their migration and lifespan are attractive approaches in treatment strategies of bovine respiratory dis-ease. Nanotechnology holds significant potential to design new compounds by our ability to manipulate at the nanoscale. Helical rosette nanotubes are a class of novel, biologi-cally inspired, water soluble and metal-free nanotubes. I used helical rosette nanotubes conjugated to arginine-glycine-aspartic acid (RGD-RNT) to study their effects on neu-trophil chemotaxis, cell signaling and apoptosis. Bovine neutrophils exposed to 5% RGD-RNT reduced their migration in response to fMLP (formyl-Methionyl-Leucyl-Phenylalanine), compared to the non-treated group (P<0.001). This inhibitory effect was the same as that of groups treated with ERK1/2 inhibitor (UO126) and p38 MAPK in-hibitor (SB239063). In addition, the phosphorylated ERK1/2 and p38 MAPK for the first time were quantified by sandwich ELISA to elucidate the mechanism of neutrophil mi-gration. The phosphorylation of both the ERK1/2 and p38 was inhibited at 5 minutes by RGD-rosette nanotubes (P<0.05). Furthermore, integrin ÑvÒ3 is possibly involved in mi-gration of bovine neutrophils. Moreover, RGD-RNT did not induce apoptosis of bovine neutrophils which was inversed by pre-exposing them to LPS for 30 minutes (P<0.001). These experiments provide the first evidence that RGD-rosette nanotubes suppress phos-phorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils.

Neutrophil granulocytes constitute the front line of defense in the innate immune response to invading microorganisms, but can also contribute to development of inflammatory disease and tissue destruction following e.g. myocardial infarction or stroke. During inflammatory activation, neutrophils leave the blood, interact with extracellular matrix proteins, and migrate into tissues in response to chemotactic factors to phagocytose and kill infectious agents by using toxic granule contents and reactive oxygen metabolites. The functional neutrophil response relies on exocytosis of cytoplasmic granules, each containing membrane proteins, which are thereby mobilized to the plasma membrane. Specific programmed cell death (apoptotic) pathways regulate neutrophil homeostasis, where an inflammatory milieu can prolong the life span of neutrophils to several days, whereas non-activated neutrophils are committed to constitutive/spontaneous apoptosis within hours. Signal regulatory protein alpha (SIRPα) is a surface glycoprotein with two intracellular immunoreceptor-tyrosine-based inhibitory motifs (ITIMs), which is highly expressed in neutrophils and other myeloid cells. In other cell types, SIRPα has been shown to regulate cellular functions such as cell migration and phagocytosis. The aim of the present thesis was to investigate neutrophil SIRPα expression in response to inflammatory activation or apoptosis, and how this receptor can regulate neutrophil adhesion and cell migration. Neutrophils contain several subcellular granule compartments, including primary (azurophilic), secondary (specific), tertiary (gelatinase) granules, and a fourth compartment called secretory vesicles. In resting neutrophils, SIRPα was found to be present in the plasma membrane and in all types of granules except for the azurophilic granules. Stimulation with the bacterial peptide fMLF in vitro, or inflammatory activation in vivo, was found to rapidly mobilize SIRPα to the neutrophil cell surface. In mice expressing a mutated form of SIRPα, where the cytoplasmic signaling domain was deleted, we found an enhanced accumulation of neutrophils in the peritoneal cavity in a peritonitis model. These findings therefore suggest that an increased amount of SIRPα on the surface of activated neutrophils could serve to negatively fine-tune neutrophil accumulation in inflammation. Neutrophil priming means that the cell becomes partially activated, in a way that facilitates subsequent full activation. One part of the priming process is a moderate exocytosis of granules, mostly the secretory vesicles, which increases the density of certain receptors on the cell surface. It also involves the activation of adhesion receptors called integrins. We found that TNFα-induced priming involved an increased accumulation of SIRPα on the cell surface. When comparing wild-type and SIRPα-mutant neutrophils, we found a strongly reduced TNFα-stimulated and β2 integrin-dependent adhesion of mutant neutrophils to type I collagen or fibrinogen. This adhesion defect resulted in a reduced adhesion-dependent activation of the respiratory burst and an increased chemotactic response of SIRPα-mutant neutrophils in vitro. During neutrophil apoptosis, several receptors are known to be shed from the cell surface (e.g. CD16 and CD43). We found that also SIRPα is shed from the surface during spontaneous as well as Fas-induced apoptosis. The shedding mechanism was found to involve matrix metalloproteinase (MMP) activity, mostly that of MMP-3 and MMP-8. In conclusion, neutrophil cell surface SIRPα expression is regulated during neutrophil activation and seems to play an important role in stimulating β2-integrin-dependent adhesion. This way, SIRPα can negatively fine-tune neutrophil migration and accumulation in inflammation. During apoptosis, SIRPα is shed from the cell surface, which may be one mechanism contributing to the well-known down-regulation in the adhesiveness of apoptotic neutrophils.

Lėtinė obstrukcinė plaučių liga (LOPL) - lėtai progresuojanti kvėpavimo takų obstrukcija, atsirandanti dėl nenormalaus uždegiminio plaučių atsako į žalingas su oru įkvepiamas daleles ar dujas. Tai liga, lemianti didelį sergamumą ir mirtingumą visame pasaulyje. Sergant LOPL labiausiai pažeidžiami apatiniai kvėpavimo takai, o taip pat periferiniame kraujyje nustatomas uždegimo žymenų koncentracijų padidėjimas. Pastarasis rodo, jog vyksta sisteminis uždegimas. Kvėpavimo takų sekretas yra biologinė terpė, kurioje esantys imuniniai žymenys atspindi vietinį uždegimą. Tiek vietinio, tiek ir sisteminio uždegiminio proceso metu pagrindinėmis ląstelėmis įvardijamos neutrofilai. LOPL yra daugiakomponentė liga ir siekiant suprasti šios ligos metu vykstančius įvairius patogeninius mechanizmus, būtina įvertinti vietinį ir sisteminį uždegimą jungiančias jungtis bei principus. Norint nustatyti vietinio ir sisteminio uždegimo sąsajas, svarbu įvertinti kvėpavimo takų sekreto tiesioginį poveikį kraujo neutrofilų funkcijoms. Šio darbo tikslas buvo įvertinti kraujo neutrofilų chemotaktinį ir fagocitinį aktyvumą bei reaktyvių deguonies formų (ROS) susidarymo ypatumus sergant LOPL. Kraujo neutrofilų chemotaksis tirtas aktyvinant neutrofilus klasikiniu chemotaksį skatinančiu veiksniu- interleukinu-8 (IL-8), indukuotais skrepliais bei indukuotais skrepliais papildytais skirtingomis IL-8 koncentracijomis (10, 30, 100 ng/ml). Fagocitinis kraujo neutrofilų aktyvumas bei ROS susidarymas buvo tirtas... [toliau žr. visą tekstą] / Chronic obstructive pulmonary disease (COPD) - slowly progressive airway obstruction resulting from abnormal inflammatory response to lung damaging particles of inhaled air or gas. It is a major cause of high morbidity and moratlity worldwide. Patients with COPD has lung injury, but determined increased blood levels of inflammatory markers shows and systemic inflammation. Neutrophils are the key inflammatory cells both in local and systemic inflammatory process. COPD is heterogeneous disease and in order to understand a variety of pathogenic mechanisms it is necessary to evaluate local and systemic inflammation combination characteristics and principles. It is also important to investigate if airway mucus influence blood neutrophils activity. The aim of this study was to evaluate both chemotactic and phagocytic activity and reactive oxygen species (ROS) production in peripheral blood neutrophils during COPD. Blood neutrophils chemotaxis was measured by activation of the interleukin-8 (IL-8), induced sputum and induced sputum and IL-8 different concentration (10, 30 and 100 ng/ml) combination. Phagocytic activity and ROS generation of blood neutrophils was measured after neutrophils activation with serum osponized/non-osponized S. aureus bacteria, induced sputum and induced sputum combination with S. aureus bacteria. The concentrations of 10, 30 and 100 bact./neutrophil S. aureus were used for all experiments. All features of neutrophils in vitro were analyzed by flow... [to full text]

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) occurs subsequent to intravenous and oral bisphosphonate exposure in a small subset of patients. Evidence of concurrent bacterial colonization at sites of bone necrosis, previous reports of neutrophil-related complications in some patients taking bisphosphonates along with perturbed neutrophil function in bisphosphonate-treated mice suggests an innate immune role in the development of bisphosphonate-related osteonecrosis of the jaws. This study investigates neutrophil function in BRONJ patients to determine if neutrophil functional defects may serve as a potential biomarker for BRONJ susceptibility. Patients with BRONJ and patients beginning intravenous pamidronate were studied. Eighteen patients with BRONJ and five patients beginning pamidronate therapy provided oral and blood neutrophil samples. Neutrophils from the population of patients with bisphosphonate-related osteonecrosis of the jaws and from those post-pamidronate treatment showed lower reactive-oxygen species production. These data suggest that a compromise in neutrophil function may be a potential biomarker for BRONJ susceptibility.

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) occurs subsequent to intravenous and oral bisphosphonate exposure in a small subset of patients. Evidence of concurrent bacterial colonization at sites of bone necrosis, previous reports of neutrophil-related complications in some patients taking bisphosphonates along with perturbed neutrophil function in bisphosphonate-treated mice suggests an innate immune role in the development of bisphosphonate-related osteonecrosis of the jaws. This study investigates neutrophil function in BRONJ patients to determine if neutrophil functional defects may serve as a potential biomarker for BRONJ susceptibility. Patients with BRONJ and patients beginning intravenous pamidronate were studied. Eighteen patients with BRONJ and five patients beginning pamidronate therapy provided oral and blood neutrophil samples. Neutrophils from the population of patients with bisphosphonate-related osteonecrosis of the jaws and from those post-pamidronate treatment showed lower reactive-oxygen species production. These data suggest that a compromise in neutrophil function may be a potential biomarker for BRONJ susceptibility.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 原真由氏の博士論文として提出された

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Copies of author's previously published articles inserted. / Bibliography: leaves 190-225. / vi, 225 leaves, [66] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the time course and degree of neutrophil polarisation in plasma and compares this response with those induced by FMLP, purified plasma proteins (particularly immunoglobulin type G) and chemotactic imflammatory mediators. In addition, the possible roles of extracellular divalent cations (Ca2+ and Mg2+), intracellular Ca2+ ions and actin microfilament distribution during responses to each stimulus are examined. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1993

Title page, summary and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / Tumor necrosis factor (TNF) is a pro-inflammatory cytokine with multiple biological effects. The receptors for this cytokine on neutrophils have been shown to be rapidly down-regulated following activation, leading to the release of soluble forms of these receptors. Thus neutrophils become less responsive to TNF and the soluble TNF receptors (TNFR) serve to control TNF activity. During inflammation, leukocytes become activated as a result of the action of a variety of mediators. These mediators include not only cytokines but also lipids, such as the pro-inflammatory 00-6 fatty acid, arachidonic acid (AA) and its metabolites. Cellular activation leads to the release of AA from membrane phospholipids. AA regulates the function of many cell types including neutrophils. In view of the known pro-inflammatory properties of AA and the anti-inflammatory properties of 00-3 fatty acids, a study was undertaken to examine whether or not these fatty acids regulate the expression and release of TNFR in neutrophils. While much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA causes a rapid (10-20 min) and dose-dependent (0.5 to 30 uM) increase (8-fold) in the surface expression of both classes of TNFR (TNFRl and TNFRlI) on human neutrophils, at concentrations found in inflammatory fluids. This correlates with an increase in superoxide production to a TNF challenge. In contrast, both fMLP and LPS significantly reduce the expression of both TNF receptors. Interestingly, in neutrophils pretreated with AA, fMLP causes an increase in TNF receptor expression, consistent with AA preventing the fMLP-induced receptor release in neutrophil culture. In addition, while AA causes an increase in TNF receptor expression on matured HL-60 cells (neutrophil-like cells), a decrease occurs on HUVEC and non-matured HL-60 cells. These data demonstrate a unique effect of AA on neutrophils. The relationship between AA and the anti-inflammatory (0-3 fatty acids, DHA and eicosapentaenoic acid (EPA), in the modulation of TNF receptor expression has also been examined. These (0-3 polyunsaturated fatty acids, including linolenic acid (LNA), cause a decrease in TNFR expression on neutrophils. The (0-6 linoleic acid (LA) and (0-9 oleic acid (OA) both cause an increase in TNFR expression. Furthermore, pre-exposure of neutrophils to nanomolar amounts of EPA or DHA prevents the AA-induced up-regulation of TNFR. These results thus identify another mechanism of regulating the inflammatory reaction by the (0-3 fatty acids. The mechanisms by which AA induces an increase in TNFR expression have been studied. Masking of the carboxyl group results in loss of activity. It is unlikely that a product of AA is responsible since neither the hydroperoxyeicosatetraenoic acid, nor hydroxyeicosatetraenoic acid derivatives show activity. Also, the effects of AA are not sensitive to the action of inhibitors of the cyclooxygenases and lipoxygenases. Using chemical inhibitors of intracellular signaling pathways, we demonstrate that the effect of AA on TNFRI is very sensitive to GFI09203X, PD098059, AACOCF3 and wortmannin, showing a role for protein kinase C, the extracellular signal regulated protein kinases and cytoplasmic phospholipase A2, and PI-3 kinase respectively, in the enhancement of TNF receptor expression by AA. Although the effects of AA on TNFRII are also decreased by the chemical inhibitors, the results show that these signalling molecules only contribute in part to the mechanisms of increased TNFRII receptor expression. The data presented in this thesis suggest a novel role for AA in the inflammatory reaction, through its action on neutrophil TNFR expression. The work has identified a unique effect of 00-3 polyunsaturated fatty acids for regulating this AA-induced increase in the expression of TNF receptors. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1141955 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Paediatrics, 2004