26 research outputs found
<i>In Vitro</i> Inflammation Inhibition Model Based on Semi-Continuous Toll-Like Receptor Biosensing
<div><p>A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an <i>in vivo</i>-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., <i>Pseudomonas aeruginosa</i>) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-ÎşB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests.</p></div
Simulation of anti-inflammatory agent screening based on standard controls in the repetitive inhibition response curve.
<p>According to the testing scheme (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g003" target="_blank">Figure 3A</a>), the stimulation-and-restoration cycle was repetitively maintained three times without chemical treatment (no inhibition) or separately with sodium salicylate treatment (positive inhibition). When two different substances were employed in the inhibition testing, CAPE (90 µM) showed positive anti-inflammatory response (A) when compared with the two control curves, whereas acetaminophen (10 mM) revealed a negative inhibition response (B). All experiments were carried out in duplicate and comparisons to the control (No inhibition) were marked as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g002" target="_blank">Figure 2</a> legend.</p
Simulation of the drug effect persistency testing using sodium salicylate.
<p>Pre-incubation with sodium salicylate during each cycle caused consistent anti-inflammatory responses to the three repeated bacterial stimulations (“1,2,3-inhibition” as a positive control) compared to that without inhibition (Non-inhibition as negative control). However, when the treatment was skipped at either the second (“1,3-inhibition”) or third cycle (“1,2-inhibition”), the inhibitory effect was not shown at the corresponding stage. This may indicate that drug efficacy persisted for 1 day. Each measurement was repeated twice under the same conditions and the significance was indicated.</p
Mechanism of inflammatory response via NF-ÎşB activation against bacterial invasion.
<p>Inflammation proceeds mainly via two different routes, which are initiated by TLRs-PAMPs binding (Infection process) or bradykinin-BR interactions (Inflammation process). The former invokes inflammation by producing cytokines as a result of the response. NF-ÎşB activation also increases the densities of other mediators such as TLRs and BRs.</p
Application of the semi-continuous analytical approach for TLR regulation to test the anti-inflammatory substance effect.
<p>Optimal conditions for the TLR regulation scheme were determined (A) and TLR expression was observed in response to two cyclic repeated bacterial stimulations (B, No treatment). Sodium salicylate was the positive inhibitor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g002" target="_blank">Figure 2</a>) and an effective dose (50 mM) was used either by co-incubation with the stimulus agent (Co-addition) or by pre-incubation (Pre-incubation). Both treatments revealed inhibition of the TLR response to serial stimulations, suggesting a model for anti-inflammatory substance screening. Each measurement was repeated twice under identical conditions and comparisons to the control (No treatment) were marked as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g002" target="_blank">Figure 2</a> legend.</p
Toll-Like Receptor-Based Immuno-Analysis of Pathogenic Microorganisms
In this study, a novel mammalian cell receptor-based
immuno-analytical
method was developed for the detection of food-poisoning microorganisms
by employing toll-like receptors (TLRs) as sensing elements. Upon
infection with bacterium, the host cells respond by expressing TLRs,
particularly TLR1, TLR2, and TLR4, on the outer membrane surfaces.
To demonstrate the potential of using this method for detection of
foodborne bacteria, we initially selected two model sensing systems,
expression of TLR1 on a cell line, A549, for <i>Escherichia coli</i> and TLR2 on a cell line, RAW264.7, for <i>Shigella sonnei</i> (<i>S. sonnei</i>). Each TLR was detected using antibodies
specific to the respective marker. We also found that the addition
of immunoassay for the pathogen captured by the TLRs on the mammalian
cells significantly enhanced the detection capability. A dual-analytical
system for <i>S. sonnei</i> was constructed and successfully
detected an extremely low number (about 3.2 CFU per well) of the pathogenic
bacterium 5.1 h after infection. This detection time was 2.5 h earlier
than the time required for detection using the conventional immunoassay.
To endow the specificity of detection, the target bacterium was immuno-magnetically
concentrated by a factor of 50 prior to infection. This further shortened
the response to approximately 3.4 h, which was less than half of the
time needed when the conventional method was used. Such enhanced performance
could basically result from synergistic effects of bacterial dose
increase and subsequent autocrine signaling on TLRs’ up-regulation
upon infection with live bacterium. This TLR-based immuno-sensing
approach may also be expanded to monitor infection of the body, provided
scanning of the signal is feasible
The generation of mouse with uterine specific <i>Pik3ca</i> ablation.
<p>(A) The expression of <i>Pik3ca</i> mRNA was evaluated in uteri by real time PCR. The data represents the mean ± SEM. *, p < 0.05. PIK3CA protein expression was assessed by Western blot (B) and immunohistochemistry (C). Total RNA and protein were prepared from whole uteri. (D) The efficiency of PIK3CA loss was evaluated in uterus of <i>Pik3ca</i><sup><i>d/d</i></sup> mice at GD 2.5 (a and d), GD 3.5 (b and e), and GD 7.5 (c and f).</p
<i>Pik3ca</i><sup><i>d/d</i></sup> mice were subfertile.
<p><i>Pik3ca</i><sup><i>d/d</i></sup> mice were subfertile.</p
The defect of gland formation in <i>Pik3ca</i><sup><i>d/d</i></sup> mice.
<p>(A) Gross anatomy of 8-week-old control (a) and <i>Pik3ca</i><sup><i>d/d</i></sup> (b) mice. (B) There was a significant decrease in the ratio of uterine weight in <i>Pik3ca</i><sup><i>d/d</i></sup> mice. (C) Histology of the uterus of control (a) and <i>Pik3ca</i><sup><i>d/d</i></sup> (b) mice. The expression of FOXA2 in the uterus of control (c) and <i>Pik3ca</i><sup><i>d/d</i></sup> (d) mouse. (D) Quantification of FOXA2 positive endometrial glands in mouse uteri. The results represent the mean ± SEM. *, p < 0.05; ***, p < 0.001.</p
A defect of uterine gland development in <i>Pik3ca</i><sup><i>d/d</i></sup> mice at 20 days of age.
<p>(A) The expression of PIK3CA was examined during uterine development. Immunohistochemical staining of PIK3CA in the uterus of PD 14 (a), PD 20 (b), and PD 28 (c). The uterus of GD 3.5 was used a positive control (d). (B) Gross anatomy of control (a) and <i>Pik3ca</i><sup><i>d/d</i></sup> (b) mice at PD 20. (C) The weight of uteri was not different between control and <i>Pik3ca</i><sup><i>d/d</i></sup> mice. (D) Immunohistochemical staining of FOXA2 in control (a and c) and <i>Pik3ca</i><sup><i>d/d</i></sup> (b and d) mice. (E) Quantification of FOXA2 positive endometrial glands in uteri of control and <i>Pik3ca</i><sup><i>d/d</i></sup> mice at PD 20. (F) The expression of <i>Foxa2</i> and <i>Spink3</i> in the uteri of control and <i>Pik3ca</i><sup><i>d/d</i></sup> mice. The results represent the mean ± SEM. ***, p < 0.001; **, p < 0.01.</p