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Chemiluminometric immuno-analysis of innate immune response against repetitive bacterial stimulations for the same mammalian cells.

Jeon JW, Cho IH, Ha UH, Seo SK, Paek SH - Sci Rep (2014)

Bottom Line: In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder.The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum.Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio-Microsystem Technology, Korea University, 1, 5-ka, Anam-dong, Seongbuk-Gu, Seoul 136-701, Korea.

ABSTRACT
For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.

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Related in: MedlinePlus

Expression of TLRs on the cell surfaces of A549 in response to the stimulation with bacterial lysate.To select an infectious agent, three bacteria, P. aeruginosa, S. sonnei, and V. parahaemolyticus, were initially used in 100-fold dilution of each lysate stock (0.1 mg/mL protein) to compare their TLRs induction efficiencies (A). Among the TLRs, TLR1, TLR2, and TLR4 were selected and the expression levels were measured using antibodies specific to each marker. Since the TLR1 expression relative to background ((A), None) in response to the stimulation with P. aeruginosa was most sensitive, the cellular response was closely examined toward the lysate dose (B). The cell numbers in the individual cultures were monitored, using the Janus green B staining technique, to show quantitatively constant (the inset). Among the TLRs, TLR1 revealed the highest sensitivity to stimulation.
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f1: Expression of TLRs on the cell surfaces of A549 in response to the stimulation with bacterial lysate.To select an infectious agent, three bacteria, P. aeruginosa, S. sonnei, and V. parahaemolyticus, were initially used in 100-fold dilution of each lysate stock (0.1 mg/mL protein) to compare their TLRs induction efficiencies (A). Among the TLRs, TLR1, TLR2, and TLR4 were selected and the expression levels were measured using antibodies specific to each marker. Since the TLR1 expression relative to background ((A), None) in response to the stimulation with P. aeruginosa was most sensitive, the cellular response was closely examined toward the lysate dose (B). The cell numbers in the individual cultures were monitored, using the Janus green B staining technique, to show quantitatively constant (the inset). Among the TLRs, TLR1 revealed the highest sensitivity to stimulation.

Mentions: Among the TLRs (e.g., TLR1, TLR2, TLR4, TLR5, and TLR6) present on the cell membrane surfaces, we tested TLR1, TLR2, and TLR4 as sensing elements for PAMPs present on three different species of pathogens (P. aeruginos, S. sonnei, and V. parahaemolyticus). Each bacterial lysate (100-fold dilution of the stock in 0.1 mg/mL protein) was used to stimulate the pre-cultured host cells (A549) to compare the cellular responses (Figure 1A). The TLR expression levels were detected using the respective anti-TLR antibodies (refer to the inset). Comparing to the backgrounds without stimulation, TLR1 stimulated with P. aeruginos and TLR4 with S. sonnei were shown to increase about 3.4-fold and 2.1-fold, respectively. On the other hand, the TLR2 expression was relatively insignificant for all infectious agents used. The elevated level of each marker could be determined by the PAMPs contained in each bacterium, and P. aeruginosa inducing the highest signal relative to background was consequently used as infectious agent of the host cells in the rest of this study.


Chemiluminometric immuno-analysis of innate immune response against repetitive bacterial stimulations for the same mammalian cells.

Jeon JW, Cho IH, Ha UH, Seo SK, Paek SH - Sci Rep (2014)

Expression of TLRs on the cell surfaces of A549 in response to the stimulation with bacterial lysate.To select an infectious agent, three bacteria, P. aeruginosa, S. sonnei, and V. parahaemolyticus, were initially used in 100-fold dilution of each lysate stock (0.1 mg/mL protein) to compare their TLRs induction efficiencies (A). Among the TLRs, TLR1, TLR2, and TLR4 were selected and the expression levels were measured using antibodies specific to each marker. Since the TLR1 expression relative to background ((A), None) in response to the stimulation with P. aeruginosa was most sensitive, the cellular response was closely examined toward the lysate dose (B). The cell numbers in the individual cultures were monitored, using the Janus green B staining technique, to show quantitatively constant (the inset). Among the TLRs, TLR1 revealed the highest sensitivity to stimulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127502&req=5

f1: Expression of TLRs on the cell surfaces of A549 in response to the stimulation with bacterial lysate.To select an infectious agent, three bacteria, P. aeruginosa, S. sonnei, and V. parahaemolyticus, were initially used in 100-fold dilution of each lysate stock (0.1 mg/mL protein) to compare their TLRs induction efficiencies (A). Among the TLRs, TLR1, TLR2, and TLR4 were selected and the expression levels were measured using antibodies specific to each marker. Since the TLR1 expression relative to background ((A), None) in response to the stimulation with P. aeruginosa was most sensitive, the cellular response was closely examined toward the lysate dose (B). The cell numbers in the individual cultures were monitored, using the Janus green B staining technique, to show quantitatively constant (the inset). Among the TLRs, TLR1 revealed the highest sensitivity to stimulation.
Mentions: Among the TLRs (e.g., TLR1, TLR2, TLR4, TLR5, and TLR6) present on the cell membrane surfaces, we tested TLR1, TLR2, and TLR4 as sensing elements for PAMPs present on three different species of pathogens (P. aeruginos, S. sonnei, and V. parahaemolyticus). Each bacterial lysate (100-fold dilution of the stock in 0.1 mg/mL protein) was used to stimulate the pre-cultured host cells (A549) to compare the cellular responses (Figure 1A). The TLR expression levels were detected using the respective anti-TLR antibodies (refer to the inset). Comparing to the backgrounds without stimulation, TLR1 stimulated with P. aeruginos and TLR4 with S. sonnei were shown to increase about 3.4-fold and 2.1-fold, respectively. On the other hand, the TLR2 expression was relatively insignificant for all infectious agents used. The elevated level of each marker could be determined by the PAMPs contained in each bacterium, and P. aeruginosa inducing the highest signal relative to background was consequently used as infectious agent of the host cells in the rest of this study.

Bottom Line: In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder.The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum.Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio-Microsystem Technology, Korea University, 1, 5-ka, Anam-dong, Seongbuk-Gu, Seoul 136-701, Korea.

ABSTRACT
For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.

Show MeSH
Related in: MedlinePlus