A novel flow cytometric assay to quantify soluble CD14 concentration in human serum

Antal‐Szalmás, Péter; Szöllösi, I.; Lakos, Gabriella; Kiss, E.; Csípő, István; Sümegi, Andrea; Sipka, Sándor; Strijp, Jos A. G. van; Kessel, van; Szegedi, Gyula

doi:10.1002/1097-0320(20011001)45:2<115::aid-cyto1153>3.0.co;2-m

Cited by 7 publications

(8 citation statements)

References 35 publications

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“…10,14,19,45,46 A major limitation of our work may be the interference of drug therapy on our findings. Therefore, the influence of steroids and immunosuppressive therapy on the lower values of CD14þ monocytes and of sCD14 observed in this study may not be completely ruled out.…”

Section: Discussionmentioning

confidence: 99%

“…10,11 Soluble CD14 has a modulator function during immune activation inhibiting monocyte response to cellbound LPS; sCD14 also regulates interleukin (IL) production by T lymphocytes and tunes immunoglobulin (Ig) secretion by B cells. 11,[14][15][16][17] Adult patients with SLE present accelerated apoptosis and decreased survival of monocytes. 8,18 In addition, in SLE patients with active disease there is increased monocyte apoptosis compared with those with inactive disease.…”

Section: Introductionmentioning

confidence: 99%

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Reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes and normal CD14 soluble levels in juvenile systemic lupus erythematosus

Liphaus

Kiss

Carrasco

et al. 2013

Lupus

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In order to evaluate Fas and Bcl-2 expressions in CD14+ monocytes, to measure soluble CD14 serum levels and to analyze the relationships with lupus nephritis and disease activity, we enrolled 41 patients with juvenile systemic lupus erythematosus (JSLE) and 27 healthy volunteers. Disease activity was determined by SLEDAI score. Peripheral monocytes were stained for CD14, Fas and Bcl-2 molecules, and cellular expressions were determined by flow cytometry. Soluble CD14 levels were measured by a quantitative ELISA kit. JSLE patients, those with active disease and those with nephritis, presented significantly reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes compared with healthy controls. Significant inverse correlations between percentages of CD14+Fas+ cells, SLEDAI score and anti-dsDNA antibodies were observed. JSLE patients had soluble CD14 levels similar to controls, although sCD14 levels positively correlated with ESR, but not with SLEDAI score. JSLE patients with nephritis also presented sCD14 levels similar to controls. In conclusion, the reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes from JSLE patients depict that monocyte apoptotic mechanisms may be important in lupus pathogenesis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes and normal CD14 soluble levels in juvenile systemic lupus erythematosus

Liphaus

Kiss

Carrasco

et al. 2013

Lupus

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“…The kinetic profiles of soluble CD14 in BAL fluids and sera were determined using a flow cytometric assay, adapted from Antal-Szalmas et al [23]. This assay is based on the competition between membrane-bound CD14 on macrophages and soluble CD14 in the test sample for binding to anti-CD14 antibodies (mAb MIL2).…”

Section: Soluble Cd14 Quantification In Bal Fluids and Seramentioning

confidence: 99%

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Gucht

Atanasova

Barbé

et al. 2006

Microbes and Infection

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Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.

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“…A physiologically relevant concentration of 2μg/mL was chosen for CD14. 45 The results show that CD14 did not significantly alter NF-κB activity in Meg-01R cells on its own, and it was not capable of promoting uLPS EC or uLPS SM to stimulate NF-κB activity ( Figure 7A). Furthermore, LPS chemotypes did not induce any activity on their own.…”

Section: Cd14 and Ll37 Do Not Enable Lps To Stimulate Nf-κb Activitmentioning

confidence: 95%

Development and characterization of a novel, megakaryocyte NF‐κB reporter cell line for investigating inflammatory responses

Vallance

Sheard

Meng

et al. 2021

Journal of Thrombosis and Haemostasis

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An easily detectable readout in megakaryocyte cell lines will enhance inflammatory research in these cells. Here, we report the development and characterization of a novel megakaryocyte NF‐κB‐reporter cell line (Meg‐01R). Multiple inflammatory molecules modulate NF‐κB activity in Meg‐01R cells. Meg‐01R cells respond to small molecule inhibitors such as IMD0354 and C87 that are known to inhibit NF‐κB activity upon stimulation with TNFα. Abstract BackgroundBecause of the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. ObjectivesThe primary objective of this study is to develop and functionally characterize a novel megakaryocyte nuclear factor κB (NF‐κB) reporter cell line to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. MethodsA Meg‐01‐NF‐κB‐GFP‐Luc (Meg‐01R) cell line was developed by inserting a reporter NF‐κB‐GFP‐Luc cassette into normal Meg‐01 cells to produce luciferase following activation of NF‐κB to enable easy detection of pro‐inflammatory and reparative signalling. Results and conclusionsMeg‐01 and Meg‐01R cells have comparable characteristics, including the expression of both GPIbα and integrin β3. Meg‐01R cells responded to various inflammatory molecules as measured by NF‐κB‐dependent bioluminescence. For example, inflammatory molecules such as tumor necrosis factor‐α and Pam3CSK4 increased NF‐κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg‐01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg‐01R cells were able to respond to lipopolysaccharide (LPS; non‐ultrapure), although it was not able to react to ultrapure LPS because of the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterization of a novel megakaryocyte NF‐κB reporter cell line (Meg‐01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF‐κB activity.

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A novel flow cytometric assay to quantify soluble CD14 concentration in human serum

Cited by 7 publications

References 35 publications

Reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes and normal CD14 soluble levels in juvenile systemic lupus erythematosus

Reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes and normal CD14 soluble levels in juvenile systemic lupus erythematosus

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Development and characterization of a novel, megakaryocyte NF‐κB reporter cell line for investigating inflammatory responses

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