Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.

The aim of this thesis was to investigate the possible role of neutrophil granulocytes in antitumor reactions. Most of our experiments were focused on in vitro studies assessing the cytotoxic effect of mouse neutrophils on B16-F10 melanoma cells labelled with PAMPs. We put an emphasis on activation and generating a prime state of neutrophils. Moreover, a release rate of enzyme myeloperoxidase from azurophil granules was detected as a marker of neutrophil degranulation. We also attempted to attract neutrophils into tumor microenvironment using thioglycolate medium and its main compound casein.

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The development of autoinflammatory diseases is caused by the dysregulation of innate immune mechanisms. This leads to the development of spontaneous inflammation. Mice lacking adaptor protein PSTPIP2 develop chronic autoinflammatory osteomyelitis due to higher activity of neutrophil granulocytes and their increased production of IL-1β. .β. PSTPIP2 interacts with PEST phosphatases and kinase CSK. These proteins are impor- tant negative regulators of Src family kinases. In this diploma thesis, the role of Src family kinases and the role of their positive regulator phosphatase CD45 in the development of chronic autoinflammatory osteomyelitis was studied. For this purpose, a mouse model of chronic autoinflammatory osteomyelitis (CMO) lacking CD45 was used. These mice deve- lop the disease with delayed kinetics. Bone marrow cells isolated from these mice produce less IL-1β. upon silica activation and have lower phosphorylation of ERK MAP kinase. It isβ. probably caused by higher phosphorylation of the inhibitory tyrosine of Src family kinases resulting in their lower activity. The presence of different immune cell populations in the bone marrow, spleen and blood of these mice was also monitored in these mice. The re- sults of this work contribute to a better understanding of the role of Src family...

Radiation and medical drugs are used to fight head and neck cancer, but unfortunately in some cases these treatments cause development of other diseases and injuries. Osteoradionecrosis (ORN) and medical-related osteonecrosis of the jaw (MRONJ) are dreaded late complications in jaws from radiation therapy and medical drugs and cause great suffering to those affected. The full extent of ORN and MRONJ may be difficult to diagnose due to visualizing problems in quantifying boundaries of osteonecrosis and healthy tissues. Maxillofacial surgeons now use radiology and clinical appearance to differ affected bone, which may result in unprecise estimation of the area that is affected. As a possible adjuvant diagnostic procedure, visualizing osteonecrosis by examining neutrophil elastase (NE) activity in jaws was tested in patients. A newly developed positron emission tomography (PET) tracer specific for NE was used for observation and measurement in PET/CT images. An image processing software was used for visualization, segmentation, and analysis. Areas with osteonecrosis were identified in the ORN patients, but not in their entirety and all activity could not be equated with osteonecrosis as undiagnosed areas as well absorbed the tracer. Visualization of MRONJ displayed unexpectedly low activity in the diagnosed area.    The conclusion drawn from the results and the analysis is that NE activity can be found in osteonecrosis patients, but the activity itself does not provide complete information to visualize and quantify the diseased area and it cannot be equated with osteonecrosis. To verify NE activity as osteonecrosis, tissue samples from the affected area need to be collected for histological examination