Journal: International Journal of Molecular Medicine

Article Title: Hypercapnia exacerbates the disruption of the blood‑brain barrier by inducing interleukin‑1β overproduction in the blood of hypoxemic adult rats

doi: 10.3892/ijmm.2020.4604

Figure Lengend Snippet: Hypercapnia increased the expression of IL-1β in the hypoxic blood in vivo and in vitro (n=4). (A) There was an interaction effect between hypoxia treatment and hypercapnia treatment (P<0.01). (B) Simple effects analyses revealed increased IL-1β expression in the hypoxemia group ( ** P<0.01), but not in the hypercapnia group (P>0.05) compared with the Sham group. The expression levels of IL-1β were the highest in the HH group in comparison with the hypoxemia group ( ** P<0.01) and the hypercapnia group ( ** P<0.01). (C) There was an interaction effect between 0.2% O 2 treatment and 10% CO 2 treatment. (D) Simple effects analyses revealed increased IL-1β expression in the hypoxia group ( ** P<0.01), but not in the HC group (P>0.05) compared with the control group. The expression levels of IL-1β were the highest in the hypoxia + HC group compared with the hypoxia group ( ** P<0.01) and the HC group ( ** P<0.01). The concentrations of O 2 and CO 2 in the air were 21 and 0.03%, respectively. IL-1β, interleukin-1β; HH, hypercapnia + hypoxemia; ns, non‑significant; HC, high concentration of carbon dioxide.

Article Snippet: The IL-1β + IL-1Ra group was treated with IL-1β (40 ng/ml) and IL-1Ra (40 ng/ml; MedChemExpress; cat. no. HY-P7029).

Techniques: Expressing, In Vivo, In Vitro, Comparison, Control, Concentration Assay

Journal: International Journal of Molecular Medicine

Article Title: Hypercapnia exacerbates the disruption of the blood‑brain barrier by inducing interleukin‑1β overproduction in the blood of hypoxemic adult rats

doi: 10.3892/ijmm.2020.4604

Figure Lengend Snippet: IL-1β treatment increased IL-1R1 and p-IRAK-1 expression in RBECs (n=4). (A) Immunoreactive bands of IL-1R1 (80 kDa) and β-actin (42 kDa). (B) The bar graph shows increased IL-1R1 expression in the IL-1β group compared with that in the control group ( ** P<0.01). (C) Immunofluorescence images showing the expression of CD31 + RBECs (a and d, green), IL-1R1 (b and e, red), and the co-localization of IL-1R1 and RBECs (c and f). Enhanced IL-1R1 immunofluorescence was evident in the IL‑1β group compared with the control group. Scale bars (a-f): 10 μ m. (D) Immunoreactive bands of p-RIAK-1 (77 kDa), t-IRAK-1 (77 kDa) and β-actin (42 kDa). (E) The bar graph shows increased p-IRAK-1 expression in the IL-1β group compared with the control group ( ** P<0.01). The protein expression of p‑IRAK‑1 was significantly suppressed with IL‑1Ra treatment ( * P<0.05). RBECs, rat brain capillary endothelial cells; IL-1β, interleukin-1β; IL‑1R, interleukin‑1 receptor; IL‑1Ra, interleukin‑1 receptor antagonist; HC, high concentration of carbon dioxide; ns, non‑significant.

Article Snippet: The IL-1β + IL-1Ra group was treated with IL-1β (40 ng/ml) and IL-1Ra (40 ng/ml; MedChemExpress; cat. no. HY-P7029).

Techniques: Expressing, Control, Immunofluorescence, Concentration Assay

Journal: International Journal of Molecular Medicine

Article Title: Hypercapnia exacerbates the disruption of the blood‑brain barrier by inducing interleukin‑1β overproduction in the blood of hypoxemic adult rats

doi: 10.3892/ijmm.2020.4604

Figure Lengend Snippet: IL-1β treatment decreased the expression of tight junctional proteins in RBECs (n=4). (A) Immunoreactive bands of ZO-1 (220 kDa), occludin (59 kDa), claudin-5 (23 kDa) and β-actin (42 kDa). (B) There was decreased tight junctional protein expression in the IL-1β group compared with the control group (ZO-1: ** P<0.01; occludin: ** P<0.01; and claudin-5: ** P<0.01). The expression of tight junctional proteins was significantly upregulated following IL-1Ra treatment (ZO-1: ** P<0.01; occludin: ** P<0.01; and claudin-5: * P<0.05). RBECs, rat brain capillary endothelial cells; ZO-1, zonula occludens-1; IL-1β, interleukin-1β; IL‑1Ra, interleukin‑1 receptor antagonist; ns, non‑significant.

Article Snippet: The IL-1β + IL-1Ra group was treated with IL-1β (40 ng/ml) and IL-1Ra (40 ng/ml; MedChemExpress; cat. no. HY-P7029).

Techniques: Expressing, Control

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-1 receptor antagonism reveals a yin-yang relationship between NFκB and interferon signaling in chronic lymphocytic leukemia.

doi: 10.1073/pnas.2405644121

Figure Lengend Snippet: Fig. 1. Effect of anakinra on NFκB in CLL cells in vitro. (A) RNAseq was performed on CLL cells from 3 patients cultured with or without anakinra for 24 h. The enrichment plot indicates downregulation of NFκB-activated genes by anakinra. (B–D) CLL cells from untreated patients were cultured for 24 h with or without resiquimod (S) at 1 μg/ml (B) or resiquimod and IL-2 (2S) at 500 IU/ml (C and D) in the presence and absence of anakinra (K). (B) Phospho-p65 was quantified in lysates collected at the indicated times by immunoblotting and densitometry using β-actin as a loading control. A sample blot is shown above the summary graph, indicating averages and SE for 7 patient samples. The averages and SEM at each time point (S vs S + K) are 0.848±0.111 vs 1.308 ± 0.220; 1.167 ± 0.161 vs 1.177 ± 0.263; 1.586 ± 0.103 vs 1.263 ± 0.125; 1.174 ± 0.174 vs 0.822 ± 0.133; 1.235 ± 0.159 vs 0.702 ± 0.163; 1.087 ± 0.075 vs 0.484 ± 0.105. (C) The lines (N = 10) indicate TNFα in culture supernatants measured by the ELISA for single patient samples. (D) Cell surface CD83 was measured by flow cytometry (N = 6). Examples of histograms are shown, with the numbers representing differences between percentages of cells stained with CD83 antibody (red histogram) or control antibodies (gray histogram). (E and F) p105/50 and p100/52 were measured in CLL cells cultured alone (E) or with TNFα (10 ng/ml) or trimeric CD40L (1 μg/ml) to activate canonical and noncanonical NFκB, respectively (F) with or without anakinra. Examples are shown. Averages and SE for (E) are p105: 0.948 ± 0.146 vs 0.821 ± 0.119, p50: 1.187 ± 0.125 vs 0.947 ± 0.105 (N = 19), p100: 0.6 ± 0.121 vs 0.355 ± 0.132, and p52: 0.404 ± 0.121 vs 0.48 ± 0.096 (N = 16) and for (F) are p105: 1.090 ± 0.203 vs 0.824 ± 0.198, p50: 1.125 ± 0.186 vs 0.713 ± 0.138 (N = 6), p100: 0.934 ± 0.182 vs 0.980 ± 0.121, and p52: 1.458 ± 0.176 vs 1.158 ± 0.186 (N = 8). Each closed circle in the summary graphs indicates results for a single sample. Statistical significance was determined with Student’s t tests and one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: They were then stained overnight at 4 °C with primary mouse anti- human IL- 1Ra antibodies (R&D systems, catalog no. MAB280) (1:500 in 5% goat serum (Gibco, Cat. 16210072) in PBS).

Techniques: In Vitro, Cell Culture, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-1 receptor antagonism reveals a yin-yang relationship between NFκB and interferon signaling in chronic lymphocytic leukemia.

doi: 10.1073/pnas.2405644121

Figure Lengend Snippet: Fig. 2. Localization of anakinra and effect on oxidative stress in CLL cells. (A) IL-1α (1.24 ± 1.006) (N = 3 0), IL-1β (0.997 ± 0.158) (N = 34), and IL-1Ra (98.475±28.467) (N = 28) were measured in supernatants of CLL cells after 48 h. Patient numbers differ because samples were assayed in different batch- es. Each circle is the result for an individ- ual sample. Averages and SE are shown. (B) CLL cells were stimulated with IL-1β or resiquimod (S) (N = 3). Phospho-p38 was quantified after 30 min by immunoblotting and densitometry with β-actin as a loading control. A representative blot is shown. In- dividual patient samples in the graph are indicated by dots. Averages and SE are Con: 0.33 ± 0.201, IL-1β: 0.264 ± 0.003, and S: 3.497 ± 0.458. (C) CLL cells precultured with or without neutralizing IL-1 receptor antibodies for 2 h before adding IL-2 and resiquimod (2S) in the presence or absence of anakinra (K) for 48 h were analyzed by confocal microscopy as described in the materials and methods. Representative images are shown, with similar results ob- tained with 5 different patient samples. (D) IL-1Ra-staining quantified for 5 images per treatment per patient sample is shown in the summary graph, with each dot repre- senting a different image. Averages and SE of mean fluorescence intensities (MFIs) are Con: 91.407 ± 3.589, 2S:169.692 ± 38.945, 2S+K:1263.622 ± 145.864, and 2S+K + anti- IL-1RI: 1741.328 ± 208.275. (E) CLL cells (N = 7) were cultured with or without anak- inra (K) for 48 h and stained with DCFH to measure intracellular ROS levels. Rep- resentative histograms (Left) and MFIs of DCFH staining for individual patient samples (Right) are shown. Statistical significance was determined using Student’s t tests. *P < 0.05; **P < 0.01; ns, not significant.

Article Snippet: They were then stained overnight at 4 °C with primary mouse anti- human IL- 1Ra antibodies (R&D systems, catalog no. MAB280) (1:500 in 5% goat serum (Gibco, Cat. 16210072) in PBS).

Techniques: Western Blot, Control, Confocal Microscopy, Staining, Fluorescence, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-1 receptor antagonism reveals a yin-yang relationship between NFκB and interferon signaling in chronic lymphocytic leukemia.

doi: 10.1073/pnas.2405644121

Figure Lengend Snippet: Fig. 4. Effect of anakinra on IFN responses in CLL cells in vivo and in vitro. (A) DEGs in circulating CLL cells from six patients at C1D1 and C1D14 were compared by GSEA. The enrichment plot indicates that anakinra up-regulated IFN-stimulated gene expression. (B) Phospho-STAT2 was measured in protein extracts from cryopreserved CLL cells obtained at C1D1 and C1D14 (N = 5) by immunoblotting and densitometry using β-actin as a loading control. (C and D) CLL cells were cultured with and without anakinra (K). Phospho-STAT2 (exposure time 1 min) was measured after 24 h (N = 11) (C). CXCL10 was measured by the ELISA after 48 h (N = 10) (D). (E–G) CLL cells were cultured with or without anakinra for 24 h and then stimulated with IFNβ. E. OASL transcripts were measured after 4 h (N = 11). Averages and SE are Con: 0.219 ± 0.076, K: 0.386 ± 0.117, IFNβ: 7.41 ± 1.617, K + IFNβ: 10.115 ± 2.041. (F) Phospho-STAT1 and -STAT2 (exposure time 5 s) were measured after 30 min (N = 7). Averages and SE are Con: 0.09 ± 0.032, IFNβ: 1.271 ± 0.197, K: 0.098 ± 0.035, K + IFNβ: 2.348 ± 0.244 for P-STAT1 and Con: 0.063 ± 0.024, IFNβ: 1.161 ± 0.254, K: 0.058 ± 0.018, K + IFNβ: 2.395 ± 0.456 for P-STAT2. (G) LAG3 was measured after 24 h. A representative example is shown in Supplementary Fig. S6. Percentages of LAG3+ cells in each condition normalized to the result for IFN-treated cells without anakinra are plotted in the summary graph. Examples of immunoblots are shown, and each line or dot in the graphs represents results for an individual patient. The means and SEMs normalized to IFNβ are Con: 0.251 ± 0.045, K: 0.327 ± 0.117, IFNβ: 1 ± 0, and IFNβ+K: 1.183 ± 0.045. (H and I) CLL cells were cultured in RPMI-1640 in the absence (H) or presence (I) of IFNβ with or without K, anifrolumab (ANI), or both (N = 14). Live (DAPI−) cells were measured after 4 d. Examples of contour plots are shown, with numbers indicating % DAPI− cells. Each dot in the summary graphs represents an individual patient. (H) Averages and SE of % live cells are Con: 29.666 ± 1.591, K: 38.369 ± 2.546, ANI: 30.65 ± 1.733, and K + ANI: 31.428 ± 2.104. (I) Averages and SE are Con: 60.839 ± 5.642, K + IFNβ: 78.786 ± 4.617, K + IFNβ+ANI: 51.804 ± 6.57. Statistical analysis was performed with Student’s t tests and one-way ANOVAs. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant.

Article Snippet: They were then stained overnight at 4 °C with primary mouse anti- human IL- 1Ra antibodies (R&D systems, catalog no. MAB280) (1:500 in 5% goat serum (Gibco, Cat. 16210072) in PBS).

Techniques: In Vivo, In Vitro, Gene Expression, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent 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: (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