Journal: Molecular pain

Article Title: Rapid cleavage of IL-1β in DRG neurons produces tissue injury-induced pain hypersensitivity.

doi: 10.1177/17448069241285357

Figure Lengend Snippet: Figure 3. (a) The plantar incision induced a reduction in mechanical threshold against von Frey stimulation. Intrathecal IL-1RA administration significantly inhibited the reduction of the mechanical threshold for 6 h after the treatment. (b) The plantar incision induced a reduction in latency time against thermal stimulation. Intrathecal IL-1RA administration significantly inhibited reduction in the thermal pain latency for 6 h after the treatment. Data were analyzed by repeated measures two-way ANOVA, with Bonferroni’s multiple comparison test comparing pre- and post-treatment time points for each group (#p < .05, ##p < .01, ###p < .001), and Bonferroni’s multiple comparison test comparing both groups at each time point (*p < .05, **p < .01, ***p < .001). The data in the graphs are presented as the mean ± SEM (n = 6 in each group). i.t.: intrathecal administration. Intrathecal administration of IL-1β induced reduction of the mechanical threshold against von Frey stimulation (c) and reduction of latency time against thermal stimulation (d). Data were analyzed by repeated measures two-way ANOVA, with Bonferroni’s multiple comparison test comparing pre- and post-treatment time points for each group (#p < .05, ##p < .01) and Bonferroni’s multiple comparison test comparing both groups at each time point (*p < .05, **p < .01). The data in the graphs are presented as the mean ± SEM (n = 6 in each group).

Article Snippet: Mice were randomly assigned to saline and IL1 RA (500 ng/5 μL, diluted in saline, Recombinant Human IL-1ra/IL-1F3, #280-RA/CF, R&D Systems, Inc. Minneapolis, MN 55413) groups (n = 6 per group).

Techniques: Comparison

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: (a) peptides sequences of IL-1ra-ELP fusion protein (Mw=54,044 Da). The IL-1ra-ELP fusion protein was produced by bacterial expression in E. coli. (b) X-ray crystallography of IL-1ra with the lysines highlighted in magenta. (c) An image of six-well tissue culture polystyrene plates with a 5 nm Cr adhesion layer and a 45 nm Au layer evaporated yielding optically transparent gold-coated slides.

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Produced, Expressing

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: (a) Bioactivity of IL-1ra-ELP fusion protein and commercial rhIL-1ra using the inhibition assay of IL-1β stimulated RPMI 1788 cells. The percentage of inhibited proliferation is plotted as a function of excess molar IL-1ra added in 10−2–104. The amount of inhibitor, IL-1ra, for inhibition of stimulatory effect of IL-1β is determined based on the 4-parameter sigmoidal fitting curve. EC50 of IL-1ra-ELP = 211.21 ± 0.31 pM (R2= 0.92) and EC50 Commercial rhIL-1ra = 20.21 ± 0.85 pM (R2= 0.97) Data presented as mean ± SE (n=4). Inset: standard curve of IL-1β induction of RPMI 1788 proliferation.

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Inhibition

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: The amount of IL-1ra-ELP detected from SAM(a), Imm IL-1ra(b) and Sol IL-1ra(c) in the absence of THP-1 monocytes and secreted IL-1ra from the Imm IL-1ra(d) and Sol IL-1ra(e) in the presence of THP-1 monocytes over time course as detected using the LINCO plex assay. No IL-1ra on the Imm IL-1ra in the absence of THP-1 monocytes was detected over the time course tested up to 5 days showing the immobilized IL-1ra-ELP is stable on the SAM surfaces. Severed IL-1ra-ELP is the IL-1ra-ELP detached from the surface possibly due to unstable chemical coupling or enzymatic action from the monoctyes. Secreted IL-1ra is the IL-1ra secreted from THP-1 monocytes responding to the external stimuli. Added IL-1ra-ELP is the soluble IL-1ra-ELP directly added into the media. * The concentration of IL-1ra at 1 hr was close to the maximun detection limit of LINCOplex assay. Sol IL-1ra w/o THP-1 was tested up to 3 days and Sol IL-1ra with THP-1 was tested up to 5 days. N.D. not detected.

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Plex Assay, Concentration Assay

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: Schematic illustration of human THP-1 monocytes interfering with the four experimental conditions used in the cell culture experiments. i.e. (a) MHA SAMs (SAM), (b) SAMs with immobilized BSA, (c) SAMs with immobilized IL-1ra-ELP (Imm IL1-ra), and (d) MHA SAMs with soluble IL-1ra (Sol IL-1ra), each of which were seeded with either presence or absence of 1 μg/ml of LPS. LPS added to the culture well at the time when the THP-1 monocytes were seeded.

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Cell Culture

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: (a–d) phase contrast images of THP-1 monocytes to show the effect of immobilized and soluble IL-1ra-ELP fusion protein on THP-1 differentiation. Cell images cultured on (a) MHA SAM without LPS stimulation, (b) immobilized IL-1ra-ELP on the MHA SAM surface with LPS stimulation, (c) soluble IL-1ra-ELP (1 μg/ml) was directly added into the media with LPS stimulation and (d) MHA SAM with LPS stimulation, were collected 72 h after seeding. (a–c) all possessed a rounded, undifferentiated morphology. Cells circled in (d) indicate a flattened, differentiated morphology of THP-1 cells. Magnification 200X.

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Cell Culture

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: Qualitative comparison of the relative expression levels of the 10 cytokines assayed at 72hrs under the four experimental conditions with and without LPS stimulation. * The level of IL-1β was tested using ELISA assay because of detection limit of Lincoplex. N.D. is non-detectable, i.e. zero. “+” , “ ++ ” and “ +++ ” mean relatively l ow, moderate and high, respectively. “ − ” means detected less than 50 pg/ml for IL-1β, IL-4, IL-6, IL-8, TNF-α, IFN-γ, MCP-1 and IL-1ra and 100 pg/ml for MIP-1α and VEGF

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Comparison, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal:

Article Title: The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes

doi: 10.1016/j.biomaterials.2007.04.010

Figure Lengend Snippet: Bar graphs of expression levels of IL-1β, TNF-α, IL-6, IL-8, MIP-1α, MCP-1, VEGF and IL-1ra at 1, 6, 24, 48 and 72 hrs from LPS-stimulated monocytes on MHA SAMs, SAMs with immobilized IL-1ra-ELP, and MHA SAMs with soluble IL-1ra-ELP as detected using the LINCOplex assay. IL-1β expression under the same conditions as measured by ELISA due to detection limit of the LINCOplex. * Significant relative to the SAM; p<0.05

Article Snippet: The bioactivity of IL-1ra-ELP was benchmarked against commercially available human recombinant IL-1ra (rhIL-1ra) (R&D Systems, Minneapolis, MN) by IL-1ra inhibition of IL-1 induced proliferation of RPMI 1788 cells [ 28 , 29 ] using ATP production as the cell viability assay.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 1 Effect of IL1B on mbIL1R, mbIL1R2 and IL1RA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A) or with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (B). Cells were recovered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by western blot. a-Tubulin was also probed on the same membranes to ensure equal protein loading. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (C and D). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data are from three different experiments. MM, minimal medium; mb, membrane-bound; s, soluble.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control, Incubation, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 2 Effect of IL1B on sIL1R2 release and IL1RA secretion by KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A and B) or with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (C and D). The cultures supernatants were recovered to evaluate sIL1R2 release (A and C) and IL1RA secretion (B and D) by ELISA. Data were expressed as % of control (ratio of sIL1R2 or IL1RA concentrations detected in the presence of IL1 to those detected in the control basal culture medium for an equivalent period of time). *P , 0.05, **P , 0.01 and ***P , 0.001 as compared with control. Data are from three different experiments.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 3 Immunocytofluorescence of IL1R1, IL1R2 and IL1RA in KLE cells. Cells cultured in chamber slides were incubated for 24 h with the culture medium alone or containing 0.1 ng/ml IL1B. Detection of IL1R1 (A and C), IL1R2 (E and G) and IL1RA (I and K) was performed by immunocytochemical staining using specific monoclonal mouse antibodies. Note the increase in IL1R1 (C), IL1R2 (G) and IL1RA (K) immunofluorescent signal in KLE cells exposed to IL1B by comparison with non-stimulated cells (A, E and I, respectively). No immunofluorescence was observed in the absence of primary antibodies (B, F and J) or the presence of mouse IgGs (D, H and L) (controls). Data are representative of four different experiments.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Cell Culture, Incubation, Staining, Comparison

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 4 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A, B and C, respectively) or with different con- centrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (D, E and F, respectively). Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1, IL1R2 and IL1RA mRNA levels were normalized to GAPDH mRNA levels. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of time). *P , 0.05, **P , 0.01 and ***P , 0.001 as compared with control. Data are from three different experiments.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 5 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA stability and gene expression in KLE cells. Confluent KLE cell cultures were stimulated with IL1B (1 ng/mll) for 12 h. Actinomycin D (10 mg/ml) was added to stop the de novo RNA synthesis, and cells were harvested after 0, 2, 4 and 8 h of incubation with actinomycin D. Total RNA was extracted and reverse transcribed. cDNA was analyzed by real-time PCR with specific primers for IL1R1, IL1R2, IL1RA and GAPDH. Levels of IL1R1, IL1R2 and IL1RA mRNA were normalized to those of GAPDH to assess the kinetics of IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA degradation. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found at different periods of time following the arrest of de novo mRNA transcription to that found at the time of arrest); data are from three different experiments. To evaluate IL1R1, IL1R2 and IL1RA transcriptional activation (D), confluent KLE cell cultures were stimulated with IL1B (1 ng/ml) for 6 h, cell nuclei were isolated and nuclear mRNA transcription was analyzed by nuclear run-on as described in Materials and Methods. DNA samples immobilized onto nylon membranes were as follows: lane 1, IL1R1 cDNA; lane 2, IL1R2 cDNA; lane 3, IL1RA; lane 4, 28S cDNA; and lane 5, pBluescript plasmid DNA. Radioactive transcripts were from KLE cells stimulated in MM or with 1 ng/ml IL1B. Data are repre- sentative of four different experiments.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Gene Expression, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Activation Assay, Isolation, Plasmid Preparation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 6 Effect of IL1B on mbIL1R1, mbIL1R2 and IL1RA expression in endometrial epithelial cells. Confluent endometrial epithelial cell cultures were treated with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Cells were recov- ered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by Western blot (A). a-Tubulin was probed on the same membranes to ensure equal protein loading. Culture supernatants were recovered to evaluate sIL1R2 release (C) and IL1RA secretion (D) by ELISA; data were from four different endometrial samples and expressed as % of control (ratio of sIL1R2 or IL1RA concentrations detected in the presence of IL1 to those detected in the control basal culture medium for an equivalent period of 12 h). *P , 0.05, as compared with control. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (B). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data were from three different endometrial samples. sIL1R1 and sIL1R2 release and IL1RA secretion from endometrial epithelial cell cultures was also analyzed by metabolic labeling with 35S-cysteine and immunoprecipitation using a goat anti-IL1R1, goat anti-IL1R2 or rabbit anti- IL1RA antibody (E). Immunoprecipitated proteins were then analyzed by SDS-PAGE as described in Materials and Methods (representative data from three different endometrial samples); mb, membrane-bound; s, soluble.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Labeling, Immunoprecipitation, SDS Page, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 7 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in endometrial epithelial cells. Confluent cultures were treated with 0.1 ng/ml IL1B with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA levels were normalized to GAPDH mRNA levels. Data were from three different endometrial samples and expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of 12 h). *P , 0.05 and **P , 0.01 as compared with control.

Article Snippet: The optical density was determined at 450 nm, and IL1R1, IL1R2 and IL1RA concentrations were calculated by interpolation standard curve using recombinant human (rh) sIL1R1, sIL1R2 (R&D Systems) and IL1RA (GenWay Biotech Inc., San Diego, CA, USA).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation