Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single‐cell level in whole blood

Brunialti, Milena Karina Coló; Kallás, Esper G.; Freudenberg, Marina A.; Galanos, Chris; Salomão, Reinaldo

doi:10.1002/cyto.10049

Cited by 27 publications

(16 citation statements)

References 21 publications

(21 reference statements)

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“…In order to evaluate basal activation of circulating monocytes, cell surface HLA-DR and CD-11b levels were assessed by flow cytometry, as described elsewhere [13]. Monocytes were identified by monoclonal antibodies anti-CD14-pernidin chlorophyll protein (PerCP); neutrophils were excluded with CD66b-fluorescein isothiocyanate (FITC) labeling.…”

Section: Methodsmentioning

confidence: 99%

“…Intracellular production of IL-1 α , IL-6, and TNF- α was quantified by flow cytometry in monocytes stimulated with LPS and treated with monensin in order to prevent cytokine release [13]. Monocytes were identified by monoclonal antibodies for surface staining (anti-CD66b-FITC and anti-CD14-PerCP) and cytokine production was detected using monoclonal antibodies anti-IL-1 α -PE, anti-IL-6-PE, and anti-TNF α -APC or their respective isotype controls.…”

Section: Methodsmentioning

confidence: 99%

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Mild Systemic Oxidative Stress in the Subclinical Stage of Alzheimer’s Disease

Giavarotti

Simon

Azzalis

et al. 2013

Oxidative Medicine and Cellular Longevity

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Alzheimer's disease (AD) is a late-onset, progressive degenerative disorder that affects mainly the judgment, emotional stability, and memory domains. AD is the outcome of a complex interaction among several factors which are not fully understood yet; nevertheless, it is clear that oxidative stress and inflammatory pathways are among these factors. 65 elderly subjects (42 cognitively intact and 23 with probable Alzheimer's disease) were selected for this study. We evaluated erythrocyte activities of superoxide dismutase, catalase, and glutathione peroxidase as well as plasma levels of total glutathione, α-tocopherol, β-carotene, lycopene, and coenzyme Q10. These antioxidant parameters were confronted with plasmatic levels of protein and lipid oxidation products. Additionally, we measured basal expression of monocyte HLA-DR and CD-11b, as well as monocyte production of cytokines IL1-α, IL-6, and TNF-α. AD patients presented lower plasmatic levels of α-tocopherol when compared to control ones and also higher basal monocyte HLA-DR expression associated with higher IL-1α production when stimulated by LPS. These findings support the inflammatory theory of AD and point out that this disease is associated with a higher basal activation of circulating monocytes that may be a result of α-tocopherol stock depletion.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mild Systemic Oxidative Stress in the Subclinical Stage of Alzheimer’s Disease

Giavarotti

Simon

Azzalis

et al. 2013

Oxidative Medicine and Cellular Longevity

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“…We have previously shown that high concentrations of LPS do not affect the detection of CD14 with clone MfP9 used in this experiment. 22 The expression of surface activation markers or LPS binding and TNF-a-producing cells was assessed from dot-plots or histograms drawn by CellQuest software (BDIS).…”

Section: Events Acquisitionmentioning

confidence: 99%

Lipopolysaccharide—cell interaction and induced cellular activation in whole blood of septic patients

Salomão

Brunialti

Kallás

et al. 2002

Journal of Endotoxin Research

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We used biotinylated LPS (LPSb) and flow cytometry to study LPS-monocyte interaction and LPS-induced cellular activation in whole blood from septic patients (SP). Expression of surface activation markers was evaluated on monocytes (HLA-DR) and T lymphocytes (CD69 and CD95), and intracellular TNF-alpha on monocytes. Saturating curve and kinetics of LPSb detection on monocytes were similar in SP and healthy volunteers (HV). LPSb bound to monocytes was detected after 5 min of incubation in both groups, with a more pronounced decay in SP. Monocytes from SP had a lower expression of HLA-DR as compared to HV, both constitutive and upon LPS stimulation. The proportion of monocytes producing TNF-alpha after LPS stimulus was higher in HV than SP (mean +/- SD = 25.2 +/- 14.2% and 2.2 +/- 2.6%, respectively, P < 0.001). LPS-induced CD69 on T CD8+ and CD8- lymphocytes was similar for patients and controls. Expression of CD95 on T lymphocytes was higher in SP as compared to HV on T CD8+ cells (GMFI, mean +/- SD = 22.3 +/- 14.6 and 8.6 +/- 5.0, respectively, P = 0.01) and CD8- cells (GMFI, mean +/- SD = 28.3 +/- 7.7 and 14 +/- 4.3 respectively, P < 0.001). Thus, monocytes and lymphocytes seem to respond differently to LPS in septic patients. Monocyte hyporesponsiveness appears not to be related to a decreased binding capacity of LPS, but rather to an impaired signal transduction.

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“…This work was conducted using a human whole blood assay and a mouse RAW264.7 cell line. Although several systems are available for evaluating LPS-cell interactions using isolated mononuclear cells (Brunialti et al, 2002;Foss et al, 2007), cell activation inevitably occurs during the cell isolation procedure (Meier et al, 2003). This problem was circumvented in the current study through the use of a human whole blood assay, ensuring detection specificity.…”

Section: Discussionmentioning

confidence: 96%

Fumigaclavine C inhibits tumor necrosis factor α production via suppression of toll-like receptor 4 and nuclear factor κB activation in macrophages

Cao

et al. 2011

Life Sciences

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Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single‐cell level in whole blood

Cited by 27 publications

References 21 publications

Mild Systemic Oxidative Stress in the Subclinical Stage of Alzheimer’s Disease

Mild Systemic Oxidative Stress in the Subclinical Stage of Alzheimer’s Disease

Lipopolysaccharide—cell interaction and induced cellular activation in whole blood of septic patients

Fumigaclavine C inhibits tumor necrosis factor α production via suppression of toll-like receptor 4 and nuclear factor κB activation in macrophages

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