Imaging of Neutrophils and Neutrophil Extracellular Traps (NETs) with Intravital (In Vivo) Microscopy
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Neutrophil Extracellular Traps
Intravital microscopy
Histones are important structural elements of nuclear chromatin. Extracellular histones are released during cell death and the formation of neutrophil extracellular traps (NETs) by immune cells. Extracellular histones are toxic to cells and have multiple effects in inflammation, injury and coagulation. Extracellular histonesincrease in sepsis and further facilitate disorders of inflammation, tissue injury and organ dysfunction. This study aims to review the toxic effects of extracellular histones and their roles in sepsis.
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Extracellular histones; Neutrophil extracellular traps; Sepsis; Inflammation; Injury
Neutrophil Extracellular Traps
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Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.
Intravital microscopy
Functional Imaging
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When pathogens pass the physical barriers, neutrophils are the first cells that infiltrate the infected tissue. Subsequently they use several well-known strategies to destroy an invading microorganism such as phagocytosis and degranulation. Recently it was shown that neutrophils are capable of releasing their nuclear DNA in the form of an extracellular trap, know as a neutrophil extracellular traps (NETs). On the one hand, extracellular traps plays crucial role during antimicrobial defense, on the other they can cause autoimmune disorders. Therefore, rigorous quantification of ETs-DNA seems to be important both for basic research and clinical applications. This paper assesses quantification methods which were used to estimate extracellular traps formation.
Neutrophil Extracellular Traps
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Abstract Neutrophil extracellular traps are potent antimicrobial weapons; however, their formation during sterile inflammation is detrimental, and the mechanism of induction is still unclear. Since advanced age is the primary clinical risk factor for poor outcomes in inflammatory diseases, we hypothesized that sterile stimuli, represented by mitochondria, would induce neutrophil extracellular trap formation in an age-dependent manner. Therefore, we analyzed induction of neutrophil extracellular traps in patients grouped according to age or immune status and observed that neutrophils from elderly patients responded to the presence of mitochondria with enhanced neutrophil extracellular trap formation. These neutrophil extracellular traps were also found to be more oxidized and exhibited higher resistance to DNase I degradation. Additionally, a higher concentration of residual neutrophil extracellular traps was detected in the plasma of the elderly. This plasma was capable of priming neutrophils through TLR9-mediated signaling, leading to further neutrophil extracellular trap formation, which was successfully inhibited with chloroquine. Finally, in a mouse model of mitochondria-induced acute lung injury, we observed that neutrophils from aged mice displayed impaired chemotactic activity but exhibited a trend of higher neutrophil extracellular trap formation. Thus, we propose that residual neutrophil extracellular traps circulating in the elderly preactivate neutrophils, making them more prone to enhanced neutrophil extracellular trap formation when exposed to mitochondria during sterile inflammation. Further investigation is needed to determine whether this vicious circle could be a suitable therapeutic target.
Neutrophil Extracellular Traps
TLR9
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Toxoplasma gondii is known to develop extracellular traps from neutrophils in some animal species such as mice, cattle, sheep, cats, and donkeys. This study aimed to investigate the extracellular trap structures formed in dog polymorphonuclear leukocytes (PMNs) to T. gondii tachyzoites in vitro. Dog PMN was isolated using Percoll dilutions (45%, 54%, 63%, and 72%). After incubation with tachyzoites, the extracellular traps originating from the dog PMNs were observed in the extracellular areas. Histones (H3), myeloperoxidase (MPO), and neutrophil elastase (NE), the characteristic features of NETosis reaction, were detected in extracellular areas. The tachyzoites were observed between the extracellular trap structures. A positive correlation was detected between the parasite concentration and extracellular traps formation but they were not statistically significant (P > 0.05). Time-dependent relationships were also not statistically significant (P > 0.05). The extracellular traps released in the PMN-tachyzoite culture increased until the 60th min of incubation. Reactive oxygen species, MPO, and NE activities were observed in the PMN-tachyzoite culture during the incubation period. The development of extracellular traps against T. gondii in dog PMNs is reported for the first time in this study. However, it could not be determined whether the extracellular traps released from dog PMNs only mechanically immobilize or have some lethal effect on tachyzoites.
Neutrophil Extracellular Traps
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Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.
Intravital microscopy
Functional Imaging
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New methods are proposed for the measurement of the number of extracellular neutrophil traps in peripheral blood and mucosal secretion. Effect of microbial factors on the ability ofgranulocytes to release nuclear DNA into extracellular space and form extracellular traps is evaluated. Normal microflora species are shown to more actively stimulate build-up of extracellular network. Formation of extracellular neutrophil traps may be a major efficacious mechanism for mucosal antimicrobial protection.
Neutrophil Extracellular Traps
Extracellular polysaccharide
Extracellular polymeric substance
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To study the influence of Solcotichovac vaccine on formation of neutrophil extracellular traps.Solcotrichovac vaccine administered in various doses was used in order to assess the formation of neutrophil extracellular traps in peripheral blood of women of childbearing age.Therapeutic dose of Solcotrichovac stimulated the most prominent formation of neutrophil extracellular traps.Solcotrichovac vaccine stimulated non-specific arm of mucosal immunity due to formation of neutrophil extracellular traps and it is probable that this mechanism explains the effect of this vaccine.
Neutrophil Extracellular Traps
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Neutrophil Extracellular Traps
Intravital microscopy
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Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Neutrophil Extracellular Traps
Pathogenesis
Trap (plumbing)
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