Project description:Oral health is characterized by functional oral polymorphonuclear neutrophils (oPMNs). Edentulism might be associated with a loss of oPMNs because these cells enter the oral cavity primarily through the gingival crevices. The main aim of this study was to investigate the numbers of oPMNs in rinse samples obtained from edentulous (n = 21) and dentate (n = 20) subjects. A second study aim was to investigate possible differences between oPMNs and peripheral blood polymorphonuclear neutrophils (cPMNs). Apoptosis/necrosis and cell-activation markers (CD11b, CD63 and CD66b) were analyzed using flow cytometry. Reactive oxygen species (ROS) production was determined either without stimulation (constitutive) or in response to 10 ?M phorbol myristate acetate or Fusobacterium nucleatum. The edentulous subjects presented with lower oPMN counts and higher percentages of apoptotic/necrotic oPMNs compared with dentate subjects. Furthermore, oPMNs from edentulous donors expressed low levels of all three activation markers and low constitutive ROS. In contrast, oPMNs from dentate subjects expressed high levels of all three activation markers and a higher level of constitutive ROS than cPMNs. When challenged, oPMNs from edentulous subjects showed no upregulation in ROS production, whereas oPMNs from dentate subjects retained their ability to respond to stimulation. The functional characteristics of cPMNs were comparable between edentulous and dentate subjects. This study demonstrates that despite having functional cPMNs, edentulous subjects have low oPMN numbers that are functionally impaired.
Project description:Purpose of Review:Oral health is maintained in a dynamic equilibrium between the host immunity and the oral microbiome. Oral polymorphonuclear neutrophils (oPMNs) are important innate immune cells in the oral cavity. Recent Findings:The oPMNs play a co-controlling part in the maintenance of oral equilibrium. In human saliva, the oPMNs integrity is preserved, and their function remains unaffected. In general, oPMNs are in a higher state of baseline activation compared to peripheral PMNs. However, in periodontitis, the oPMNs' activation state can result in excessive release of damaging molecules in the extracellular environment. Summary:The presence of oPMNs may unwittingly negatively impact the integrity of the oral tissues. While most of the oPMN functions occur intracellularly, release of their potent active mediators into the extracellular environment may jeopardize oral homeostasis and its integrity. The dual nature of oPMNs, both beneficial and detrimental, remains a challenging and understudied topic.
Project description:The protective mechanisms that maintain periodontal homeostasis in gingivitis and prevent periodontal tissue destruction are poorly understood. The aim of this study was to identify changes in the salivary proteome during experimental gingivitis.We used oral neutrophil quantification and whole saliva (WS) proteomics to assess changes that occur in the inflammatory and resolution phases of gingivitis in healthy individuals. Oral neutrophils and WS samples were collected and clinical parameters measured on days 0, 7, 14, 21, 28, and 35.Increased oral neutrophil recruitment and salivary cytoprotective proteins increased progressively during inflammation and decreased in resolution. Oral neutrophil numbers in gingival inflammation and resolution correlated moderately with salivary ?-globin, thioredoxin, and albumin and strongly with collagen alpha-1 and G-protein coupled receptor 98.Our results indicate that changes in salivary cytoprotective proteins in gingivitis are associated with a similar trend in oral neutrophil recruitment and clinical parameters.We found moderate to strong correlations between oral neutrophil numbers and levels of several salivary cytoprotective proteins both in the development of the inflammation and in the resolution of gingivitis. Our proteomics approach identified and relatively quantified specific cytoprotective proteins in this pilot study of experimental gingivitis; however, future and more comprehensive studies are needed to clearly identify and validate those protein biomarkers when gingivitis is active.
Project description:BACKGROUND:Sjögren's syndrome antigen B is expressed in the nucleus and surface membrane of human polymorphonuclear neutrophils and is released after cell death. However, its biological role is not clear. This study is aimed to investigate the effect of Sjögren's syndrome antigen B on human polymorphonuclear neutrophils. METHODS:Human recombinant Sjögren's syndrome antigen B (rSSB) purified from E. coli was incubated with human polymorphonuclear neutrophils as well as retinoid acid-induced granulocytic differentiated HL-60 cells, HL-60 (RA). Interleukin (IL)-8 protein production and mRNA expressions were measured by enzyme-linked immunosorbent assay and quantitative-polymerase chain reaction, respectively. Uptake of fluorescein isothiocyanate (FITC)-rSSB was assessed by flow cytometry and fluorescence microscopy. Moreover, mitogen-activated protein kinase (MAPK) pathways and nuclear factor-kappaB activation were investigated. RESULTS:Human rSSB stimulated IL-8 production from normal human neutrophils and HL-60 (RA) cells in a time- and dose-dependent manner. This IL-8-stimulated activity was blocked by chloroquine and NH4Cl, indicating that endosomal acidification is important for this effect. We found rSSB activated both MAPK pathway and nuclear factor-kappaB signaling to transcribe the IL-8 gene expression of cells. Furthermore, tumor necrosis factor-? exerted an additive effect and rSSB-anti-SSB immune complex exhibited a synergistic effect on rSSB-induced IL-8 production. CONCLUSIONS:Sjögren's syndrome antigen B might act as an endogenous danger molecule to enhance IL-8 gene expression in human polymorphonuclear neutrophils.
Project description:Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.
Project description:Human polymorphonuclear neutrophils (PMNs), purified on Ficoll-Hypaque cushions, were incubated for 5 min with calf skin acid-soluble collagen and the released superoxide anions (O2-) measured spectrophotometrically by reduction of ferricytochrome c or by chemiluminescence analysis. This collagen stimulated the release of O2- unless it had been treated with pepsin. The stimulatory activity remained in denatured collagen, was contained only in the alpha 1(I) chain and was present in the alpha 1(I)-CB 6 (CNBr-cleaved) peptide, which is C-terminal. The activity was linearly dependent on the collagen concentration up to about 200 micrograms/ml. In addition, this collagen induced a release of beta-glucuronidase and N-acetyl-beta-glucosaminidase from PMNs.
Project description:Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n=4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFM-NM-1, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFM-NM-1 and IL-1M-NM-1 and IL-1M-NM-2. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. We show that these changes in neutrophil transcriptome are most likely due to a combination of early activation of circulating neutrophils by TNFM-NM-1 and G-CSF and a mobilization of young neutrophils from the bone marrow. After LPS infusion blood was taken at t=0, t=2, t=4 and t=6 hours. Neutrophils were isolated and gene expression of these cells was assessed. T=2, t=4 and t=6 were related to t=0 as control condition
Project description:Genetic studies often use genomic DNA from whole blood cells, of which the majority are the polymorphonuclear myeloid cells. Those cells undergo dramatic change of nuclear morphology following cellular differentiation. It remains elusive if the nuclear morphological change accompanies sequence alternations from the intact genome. If such event exists, it will cause a serious problem in using such type of genomic DNA for genetic study as the sequences will not represent the intact genome in the host individuals. Using exome sequencing, we compared the coding regions between neutrophil, which is the major type of polymorphonuclear cells, and CD4+ T cell, which has an intact genome, from the same individual. The results show that exon sequences between the two cell types are essentially the same. The minor differences represented by the missed exons and base changes between the two cell types were validated to be mainly caused by experimental errors. Our study concludes that genomic DNA from whole blood cells can be safely used for genetic studies.
Project description:Mycophenolic acid (MPA) is used clinically to prevent graft rejection but may increase the risk of fungal infection. We observed that MPA enhanced the Aspergillus fumigatus-induced oxidative burst of polymorphonuclear neutrophils, but without a corresponding increase in fungal killing. Furthermore, MPA inhibited the proinflammatory cytokine response and maturation of dendritic cells.
Project description:In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils. To elucidate the influence of the blood collection technique, anticoagulants and storing temperature on isolated PMNs healthy volunteers provided blood samples with different needles and collection techniques, anticoagulants (EDTA, heparin, citrate) and storing temperatures (4, 22, 37 °C). From each blood sample PMNs were isolated and compared regarding number of PMNs and oxidative burst. The blood collection technique, anticoagulants and storing temperature had minor impact on isolated PMNs. All three tested cannulas and anticoagulants can be used to obtain blood samples for PMN isolation. For storing temperatures 37 °C should be preferred. Regarding time between the PMN isolation and the actual experiments, a time span of maximum 1 h should be targeted.