Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The epistatic interrelationships of IL-1, IL-1 receptor antagonist, and the type I IL-1 receptor.

doi: 10.4049/jimmunol.169.1.393

Figure Lengend Snippet: FIGURE 1. Murine protein isoforms of IL-1ra. A, Schematic diagram showing the relationships between the murine IL-1ra protein isoforms. Shared sequences are in black, those unique to pro-sIL-1ra (namely the signal peptide) are in gray, those unique to icIL-1ra1 (alternate exon 1) are in white, and the N-terminus that is deleted from icIL-1ra3 is hatched. B and C, Detection of the various proteins isoforms by Western blot of liver proteins from mice challenged with LPS (B) and skin proteins from un- challenged mice (C) of wt, rako, ratg, and rako, ratg mutants. The different isoforms of IL-1ra were assigned based on size relative to m.w. markers and recombinant sIL-1ra (R&D Systems), partial sequencing, and tissue distribution.

Article Snippet: ELISAs were performed as previously described and followed the manufacturers’ suggested protocols using the following Abs or kits: polyclonal goat anti-mIL-1ra (R&D Systems, Minneapolis, MN); DuoSet kits for IL-1 , IL-10, and TNF- (R&D Systems); and OptEIA for IFN- (BD PharMingen, San Diego, CA).

Techniques: Western Blot, Recombinant, Sequencing

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 1. Time course of mRNA expression of IL-1 cyto- kines and receptors produced by cultured huMCs after ag- gregation of FcεRI. Cells were exposed to anti–NP-IgE over- night, followed by the addition of antigen () to paired cell cultures. Total RNA was isolated at the indicated times and the RPA was performed. A representative result is shown (A). To establish the identity of each protected fragment, the known size and migration distance of the unprotected probe was used to prepare a standard curve. Human control RNA (BD Pharmingen) was used as a positive control and yeast tRNA was used as a negative control. Semiquantification of IL-1ra (B), IL-1RI (C), and IL-1 (D) expression in huMCs before (open bars) or after (filled bars) aggregation of FcεRI was performed by measuring the band density of the relative expression of each mRNA with respect to L32 mRNA after each background was subtracted. IL-1RII and IL-1 mRNAs were below detection levels. For IL-1ra and IL-1 the results were repeated in five separate experiments through 8 h using cells cultured from different donors. These data are pre- sented as means SEM; *P 0.05, **P 0.005, ***P 0.001 compared with 0 time. The 4-h error bar in activated cells in D was too small to diagram.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Expressing, Produced, Cell Culture, Isolation, Migration, Control, Positive Control, Negative Control

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 4. Inhibition of IL-1–induced IL-8 production by A549 epithelial cells by IL-1ra or mast-cell lysates. A549 cells were in- cubated with IL-1 at a concentration of 50 ng/ml for 16 h in the presence or absence of either rhIL-1ra (A) or huMC lysate (B). Cell-free supernates were collected and assayed for IL-8 by ELISA. The increase was downregulated by (A) increasing con- centrations of standard IL-1ra, and (B) increasing concentrations of huMC lysate.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 3. Time course of IL-1ra release from cultured huMCs af- ter FcεRI aggregation. huMCs were incubated with anti–NP-IgE overnight at 37C and washed, and NP-BSA was added to aggre- gate FcεRI (time 0). Plates were then centrifuged at the indicated time points, the supernates removed, and cells lysed. IL-1ra was measured by ELISA in both huMC lysates (open bars) and su- pernates (filled bars) of mast cells (*P 0.05, and **P 0.005 compared with time 0). Data presented are the results of three experiments performed on huMCs cultured from a single donor.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 2. IL-1ra protein in huMCs and in supernates from huMCs after aggregation of FcεRI. (A) After staining of permeabilized cells with a phycoerythrin-conjugated anti–IL-1ra mAb (thick line) or an isotype control mouse IgG1 (thin line), FACS analysis was per- formed to detect the intracellular expression of IL-1ra. (B) huMCs were incubated with anti–NP-IgE overnight at 37C and washed, and NP-BSA was added to aggregate FcεRI. Plates were then centrifuged and the supernates removed. IL-1ra was measured by ELISA in supernates of stimulated (filled bar) and unstimulated (open bar) cells at 20 h (n 4; with cells for each experiment cul- tured from separate donors). (C) Western blot analysis of IL-1ra in huMC supernatants and lysates. ST, rhIL-1ra standard; Lys, concentrated lysate; Supp, concentrated supernate; Lys HMC-1, concentrated HMC-1 lysate. These samples were subjected to electrophoresis on 4 to 12% Bis-Tris gel under reducing condi- tions. After electrophoresis, proteins were blotted and incubated with goat antihuman IL-1ra.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Staining, Control, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Electrophoresis

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 5. IL-1ra in human lung mast cells. (A) Sequential 2-m sections of a surgically resected lung immuno- stained for tryptase (i) and IL-1ra (ii), demonstrating colocalization of IL-1ra to tryptase mast cells (filled arrows). Open arrows indicate IL- 1ra tryptase cells. (B) IL-1ra pro- duction from human lung mast cells (106 cells/ml) stimulated with FcεRI aggregation. Purified human lung– derived mast cells were incubated with anti–NP-IgE overnight at 37C and washed, and NP-BSA was added to aggregate FcεRI. Plates were then centrifuged and the supernates re- moved. IL-1ra was measured by ELISA in supernates of stimulated (filled bar) and unstimulated (open bar) cells at 20 h (n 2).

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Staining, Purification, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 6. IL-1ra levels and isoforms in BALF from seg- mental antigen challenge. Segmental antigen challenge was performed as described in MATERIALS AND METHODS, with (A) before- (Pre) and after- (Post) antigen challenge eosin- ophil number (** indicates a significant difference between eosinophil number in BALF after antigen challenge when compared with before challenge; P 0.005; (B) before- and after-challenge total cell number (* indicates a signifi- cant difference between total cell number in BALF after antigen challenge when compared with before challenge; P 0.05); and (C) IL-1ra (filled bars) and tryptase (open bars) found in cell-free supernates from BALF. Normal subjects (Normal) were 10 patients who were nonatopic and nonasthmatic. There was a significant difference (*P 0.05) between IL-1ra levels in BALF after antigen chal- lenge when compared with before-challenge and normal lev- els. (D) Western blotting of BALF shows the 17-kD form as prominent in BALF with a minimal band at 22 kD. ST, rhIL-1ra 17-kD standard; BAL 1 and 2 are representative samples of after-challenge BALF.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 1. Generation of icIL-1Ra12/2 mice. (A) Targeting strategy used for the introduction of an EGFP–Neo cassette into the icIL-1Ra1–specific first exon (icIl1rn1) of the mouse Il1rn gene. This approach does not interfere with the expression of the other transcript variants. Shown from top to bottom are the WT icIl1rn1 genomic locus (WT allele), the targeting vector and the targeted genomic locus (Mutant allele). Bold lines indicate arms of homology for recombination. Exon 1 is represented as a gray box and numbered. The neomycin resistance gene (Neor) is flanked by two loxP sites (triangles). The primers used to identify recombinant ES cells are indicated as S1 and S2. (B) Southern blot analysis of WT (+/+) and heterozygous (+/2) (one WT allele, one mutant allele) ES cells using a 59 external probe (left panel) or a 39 internal probe (right panel). The WT (6.4-kb [BglII digestion] or 8.9-kb [HindIII digestion] bands) and mutant alleles (7.4-kb [BglII digestion] or 6.2-kb [HindIII digestion] bands) were detected in BglII- or HindIII-digested ES cell genomic DNA, using, respectively, the 59 or 39 probes shown in (A). (C) PCR genotyping to distinguish WT (+/+), heterozygous (+/2) and icIL-1Ra1(2/2) mice. The primers used are indicated as P1, P2, and P3 in (A). The sizes of the WT and icIL-1Ra12/2 PCR products are 300 and 842 bp, respectively. (D) Male (left panel) and female (right panel) WT (male: n = 13/n = 6 [10/17 wk]; female: n = 11/n = 6 [10/17 wk]) and icIL-1Ra12/2 (male: n = 13/n = 13 [10/17 wk]; female: n = 11/n = 9 [10/17 wk]) mice were weighed at 10 and 17 wk of age. Data are shown as the mean 6 SEM. No significant differences in body weight were observed between the genotypes (unpaired two-tailed Student t test). (E) Analysis of IL-1Ra isoforms in lysates of BMDC generated from naive WT and icIL-1Ra12/2 (KO) mice. IL-1Ra was detected using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression. One representative sample per genotype out of n = 3 per genotype is shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms are as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa (13, 69).

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Recombinant, Southern Blot, Two Tailed Test, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 2. Characterization of BMDC and BMDM from WT and icIL-1Ra12/2 mice. (A) Determination of mRNA levels for icIL-1Ra1 (icIl1rn1) and for sIL-1Ra and icIL-1Ra3 (sIl1rn) in BMDC (left panel) and BMDM (right panel). BMDC and BMDM from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated for qRT-PCR analysis. Results represent icIl1rn1 and sIl1rn mRNA expression levels relative to Mrpl32 mRNA levels. (B) Release of IL-1Ra protein (all isoforms) into the supernatant. BMDC (left panel) and BMDM (right panel) from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra in the supernatant was measured using an ELISA that recognizes all isoforms of IL-1Ra. (A and B) Data are shown as the mean 6 SEM of values obtained from three (BMDC) or two (BMDM) independent experiments. The unpaired or paired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. (C) Analysis of IL-1Ra isoforms in lysates and cell supernatants of BMDC (left panel) and BMDM (right panel) by Western blot. BMDC and BMDM from naive WT and icIL-1Ra12/2 (KO) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra isoforms were analyzed in cell lysates or collected supernatants (30 ml) using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression in lysates. One nanogram of rsIL-1Ra served as control. Two rep- resentative samples out of n = 5 per genotype are shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms is as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa. n.d., not detected. SN, supernatant.

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Control, Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 3. icIL-1Ra1 deficiency in- creases the clinical severity of Aldara- induced ear thickness and the expression of proinflammatory cytokines. (A) IL- 1Ra isoforms were analyzed in lysates of total dorsal skin from naive WT, hetero- zygous (HET) and icIL-1Ra12/2 (KO) mice using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra iso- forms. Protein loading was assessed by the determination of GAPDH expression. One representative sample out of n = 3 per genotype is shown. Arrow indicates the icIL-1Ra1 isoform. Theoretical mo- lecular mass of icIL-1Ra1: 18 kDa. (B–E) Aldara, or Vaseline as a control, were applied on the right or left ear, respec- tively, of WT (n = 21) and icIL-1Ra12/2

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 4. icIL-1Ra1 is the main IL-1Ra isoform regulating Aldara-induced skin inflammation. (A) Representative immunohistochemical detection of IL-1Ra (brown staining) on ear sections of WT and icIL-1Ra12/2 mice on day 8 after daily application of Vaseline (control) or Aldara. Scale bar, 100 mm. (B) Analysis of IL-1Ra isoforms in lysate of Vaseline-treated (control) (upper panel) and Aldara-treated (lower panel) ears from WT and icIL-1Ra12/2

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 5. icIL-1Ra1 plays no intracellular role in keratinocytes but is released together with IL-1a upon Aldara treatment. (A) Differential gene expression analysis based on RNA-Seq data of primary keratinocytes from icIL-1Ra12/2 versus WT mice. Primary keratinocytes were isolated from WT (n = 4) and icIL-1Ra12/2 (n = 4) mouse tails and left untreated as a control (left panel) or stimulated with 100 ng/ml rec. m IL-1a (middle panel) or 100 ng/ml rec. m IL-36b (right panel) for 6 h. Total RNA was then isolated from keratinocytes for RNA-Seq analysis. A total of 10,750, 10,709, or 10,727 genes were tested for control, IL-1a–, or IL-36b–treated cells, respectively. The fold-change threshold and Benjamini–Hochberg corrected p value threshold were set to 2 and 0.05, respectively. (B) Ear explants taken from WT (n = 6) and icIL-1Ra12/2 (n = 6) mice at day 8 after daily treatment with Vaseline (control) or Aldara were cultured for 24 h. Levels of IL-1Ra, IL-1a, and IL-1b in the supernatant or the percentage of cytotoxicity were assessed by ELISA or LDH assay, respectively. The IL-1Ra ELISA recognizes all isoforms of IL-1Ra. Data are shown as the mean 6 SEM from one (Figure legend continues)

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Gene Expression, RNA Sequencing, Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal:

Article Title: Computational Design and Application of Endogenous Promoters for Transcriptionally Targeted Gene Therapy for Rheumatoid Arthritis

doi: 10.1038/mt.2009.182

Figure Lengend Snippet: Comparison of therapeutic efficacy using Saa3 versus IL1E/IL6P promoter for disease-regulated Il1rn expression. (a) Promoter activity in transduced synovium of naive and arthritic C57Bl/6 mice. Knee joints were injected with 300 ng p24gag equivalent lentivirus encoding PGK, Saa3, or IL1E/IL6P-luciferase. Seven days after transduction, arthritis was induced by intra-articular injection of 180 µg zymosan A. After 24 hours, luciferase activity was assessed ex vivo. Data are represented as individual relative luciferase activities; horizontal bars indicate the means per group. (b) Efficacy of transcriptionally targeted adenoviral vectors expressing Il1rn. NIH-3T3-5xNF-κB-Luc were transduced at a multiplicity of infection (MOI) of 10 with control vector (del) or adenovirus encoding CMV, Saa3, and IL1E/IL6P-driven Il1rn. After 24 hours, cells were transduced at an MOI of 10 with control vector (del) or Ad5.CMV-Il1b. The day thereafter, IL-1β-induced NF-κB activation was assessed by luciferase assay. Data are represented as relative luciferase activities ± SEM (n = 4), and differences were determined using analysis of variance with Dunnett's post-test. **P < 0.01.

Article Snippet: The plates were then incubated with the biotinylated goat anti-murine Il1rn (BAF 480; R&D Systems) at 0.2 μg/ml in 0.1% bovine serum albumin/PBS for 2 hours, followed by a 30-minute incubation with streptavidin-conjugated horseradish peroxidase (Dako, Glostrup, Denmark) at 0.25 μg/ml in PBS.

Techniques: Comparison, Drug discovery, Expressing, Activity Assay, Injection, Luciferase, Transduction, Ex Vivo, Infection, Control, Plasmid Preparation, Activation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Reprogramming of a subpopulation of human blood neutrophils by prolonged exposure to cytokines.

doi: 10.1038/labinvest.2009.74

Figure Lengend Snippet: Figure 8 Release of IL-1b, IL-8 and IL-1Ra by long-lived neutrophils. Freshly isolated neutrophils (bar 1) and long-lived neutrophils (bar 2) were incubated at 5 106/ml for 24 h. IL-1b (c), IL-1Ra (b) and IL-8 (a) were measured in supernatants using enzyme immunometric assays. The results are means±s.e.m. of three donors (freshly isolated neutrophils) and five donors (long-lived neutrophils). Statistics: Student’s unpaired t-test for freshly isolated neutrophils vs long-lived neutrophils (*Po0.05, ***Po0.001).

Article Snippet: Apostat intracellular caspase detection kit (FITC-VD-FMK), EIA kit for IL-1Ra (biotinylated secondary goat anti-human IL-1Ra Ab: DY280, #1144097) and Proteome Profiler: Human Phospho-MAP Kinase Array Kit were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Incubation

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced Evans blue extravasation and BBB ultrastructure. a Experimental protocol. b , c Extravasation of Evans blue dye in brain tissues of the control, vehicle, IL-1RA, and IL-1RA-PEP groups was evaluated quantitatively. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 6 per group, based on one-way ANOVA with Bonferroni correction. d Ultrastructure changes in the BBB. The basement membranes (marked between black arrows in top panels) were observed in different groups. The black frames in middle panels are areas selected for amplification in the following bottom panels, to highlight tight junctions (pointed by white arrows). Scale bar, 500 nm and 2 μm for the inserts. MCAO middle cerebral artery occlusion, BBB blood-brain barrier

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Control, Amplification

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced endogenous IgG leakage and permeability of FITC-dextran. a Extravasation of endogenous IgG, and intravenous injection and measurement of FITC-dextran tracer were evaluated in the different groups. Increases (%) in IgG ( b ) and FITC-dextran ( c ) were represented by the proportion of the areas of positive regions in the cerebral cortex of the ipsilateral hemisphere. Scale bar, 100 and 200 μm for the inserts. Data are shown as mean ± SEM; ** P < 0.01, *** P < 0.001 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 6 per group, based on one-way ANOVA with Bonferroni correction. MCAO middle cerebral artery occlusion, FITC-dextran fluorescein isothiocyanate–dextran

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Permeability, Injection

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Brain penetration effects and levels of IL-1RA and IL-1RA-PEP. a , b At 24 h after ischemia-reperfusion, the permeation effect on brain tissue was assessed by immunohistochemical staining. The rats with middle cerebral artery occlusion (MCAO) in separate groups were injected intravenously with IL-1RA or IL-1RA-PEP. c , d High magnification of double immunofluorescence images show co-localization of IL-1RA with NeuN (arrows) and IL-1RA-PEP with NeuN (arrows). Scale bar, 5.0 mm and 50 μm for the inserts. e The permeation effect was expressed as a percentage increase in staining intensity in the cerebral cortex and striatum of the contralateral hemisphere. f The concentration of IL-1RA in the ipsilateral ischemic brain hemisphere of rats subjected to MCAO, 4 h after a single intravenous injection of IL-1RA (50 mg/kg) or IL-1RA-PEP (50 mg/kg), was measured by ELISA. Panels show IL-1RA concentration in the cerebral cortex and striatum area, respectively. Data are shown as mean ± SEM; * P < 0.05 in comparison with the rat intravenously injected with IL-1RA, n = 4–6 per group, based on the Student’s t test with Welch’s correction

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced changes in tight junction protein expression and localization. a–e Protein expression levels of ZO-1, occludin, and claudin-5 in cerebral tissue of the control, vehicle, IL-1RA, and IL-1RA-PEP groups were evaluated. f–i Localization of ZO-1, occludin, and claudin-5 in the cerebral tissue of different groups was also analyzed. Scale bar = 50 μm. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 4 per group, based on one-way ANOVA with Bonferroni correction. MCAO middle cerebral artery occlusion

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced changes in MMP protein expression and localization. a–c Protein expression levels of MMP-9 and MMP-2 in cerebral tissue of the control, vehicle, IL-1RA, and IL-1RA-PEP groups were evaluated. d–f Localization of MMP-9 and MMP-2 in the cerebral tissue of different groups was also analyzed. Scale bar = 100 μm for the inserts. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 4 per group, based on one-way ANOVA with Bonferroni correction. MCAO middle cerebral artery occlusion

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on angiopoietin-1 and VEGF expression in brain tissues following ischemia-reperfusion. a–c Protein expression levels of angiopoietin-1 and vascular endothelial growth factor (VEGF) were analyzed by western blot. The gray value of the band was scanned by optical densitometry, and the ratio value was statistically analyzed. d , e The mRNA expression of angiopoietin-1 and VEGF were analyzed by real-time quantitative PCR. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group, n = 6 per group, based on one-way ANOVA with Bonferroni correction

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Role of p65/NF-κB pathways in IL-1RA-PEP-mediated effects on blood-brain barrier (BBB) disruption. a After the JSH-23 inhibitor was administered, expression levels of ZO-1, MMP-9, angiopoietin-1, and VEGF were analyzed by western blot in the different groups. b–e The gray value of the band was scanned by optical densitometry, and the ratio value was statistically analyzed. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group without JSH-23; # P < 0.05, ## P < 0.01 compared to the IL-1RA-PEP group without JSH-23; n = 4 per group, based on one-way ANOVA with Bonferroni correction

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Disruption, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Graphic summary for the effect of IL-1RA and IL-1RA-PEP on BBB disruption in rats subjected to cerebral ischemia-reperfusion injury

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Disruption

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: Western-blot analysis of IL-1ra protein in lysate and supernatant of oral mucosal cells. Lane 4 is a fraction of a concentrated pool of supernatants from three unstimulated cultures. Cell-associated IL-1ra is shown from unstimulated cells (lane 5). For comparison, lane 3 is the 25 kD monocyte IL-1ra. Lane 2 is the biotinylated standard (mol. wt. × 103 daltons). Lane 1 is the control with preimmune serum.

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Western Blot, Comparison, Control

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: IL-1-induced IL-6 secretion in OMECs, which were incubated with either RPMI (control), 20 µg/ml of neutralizing human IL-1ra (anti-IL-1ra) or recombinant IL-1α (10 ng/ml) for 24 h. IL-6 immunoreactivity in cell culture supernatants was assayed by ELISA. Exogenous IL-1 enhanced IL-6 secretion by 552%, whereas endogenous IL-1 enhanced IL-6 secretion by 261% after IL-1ra neutralization. The ‘% increase’ represents the mean of (test/control) × 100 calculated for the three experiments, each performed with different cell cultures

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Incubation, Control, Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay, Neutralization

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: Immunohistological staining in scattered oral mucosal cells (×20). These results are representative of two similar experiments. (a) IL-1ra staining is observed in a few unstimulated cells. (b) It is more intense in the cytoplasm of cells stimulated with TGF-β1. (c) These cells stain markedly positive for involucrin. (d) Staining was nil in control with preimmune serum. Control was also negative with anti-IL-1ra serum preincubated with recombinant IL-1ra.

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Staining, Control, Recombinant

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: Modulation of intracellular IL-1ra (a), IL-1β (b) and IL-1ra : IL-1β ratio (c) by cytokines in oral mucosal cells. Cells were incubated for 24 h with RPMI 1640 alone as control (C), IL-1α, TNFα, IFNγ, IL-10, TGFβ1, IL-4, IL-6, hydrocortisone (HC) or calcium. The concentration of cytokines in culture lysates was determined by ELISA. The results represent the mean of two experiments, each performed from different cultures. P < 0·05.

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Incubation, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: Modulation of intracellular IL-1ra (a), IL-1α (b) and IL-1ra : IL-1α ratio (c) by cytokines in oral mucosal cells. Cells were incubated for 24 h with RPMI 1640 alone as control (C), IFN-γ, IL-10, TGFβ1, IL-4, TNFα, IL-6 or hydrocortisone (HC). The concentration of cytokines in culture lysates was determined by ELISA. The results represent the mean of two experiments, each performed from different cultures. P < 0·05.

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Incubation, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: Summary of intracellular IL-1 family regulation in OMECs, compared with that in skin keratinocytes

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Derivative Assay

Journal:

Article Title: IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-?1 and IL-4

doi: 10.1046/j.1365-2249.2002.01685.x

Figure Lengend Snippet: Modulation of secreted IL-1ra (a), IL-1α (b) and IL-1ra : IL-1α ratio (c) by cytokines in oral mucosal cells. Cells were incubated with RPMI 1640 alone as control (C), TNFα, IL-6, IFN-γ, IL-10 or TGFβ1. The concentration of cytokines in culture supernatants was determined by ELISA. The results represent two experiments, each performed from different cultures. P < 0·05.

Article Snippet: Blots were developed using specific polyclonal antihuman IL-1ra goat antibody (R&D Systems, Abingdon, UK) at 1/100 dilution in PBS with 1% skimmed milk, biotinylated mouse antigoat secondary antibody (Sigma) at 1/100 dilution and extravidin–phosphatase complex (Sigma) at 1/100 dilution in phosphate-free buffer.

Techniques: Incubation, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay