Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Genes differentially expressed in resident macrophages at 1 h postinfection with S. pyogenes

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Zinc-Fingers, Binding Assay

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Confirmation of microarray data by RT-PCR and protein expression. (A) RT-PCR analysis of selected gene transcription in resident macrophages uninfected or infected with S. pyogenes. Uninfected samples were loaded in lanes 1, and infected samples were loaded in lanes 2. β-Actin expression served as a control. (B) IL-6 protein expression by S. pyogenes-infected macrophages. Resident macrophages were isolated from the peritoneal cavity of mice after 1 h of infection with S. pyogenes and cultured in vitro for 2 h. Noninfected macrophages were used as a control. The levels of IL-6 in the supernatants were determined by ELISA. Each column represents the mean ± standard deviation of triplicate samples obtained from three independent experiments. P < 0.0001 (ANOVA).

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Isolation, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

doi: 10.1111/j.1365-2567.2003.01762.x

Figure Lengend Snippet: Kinetics of endogenous IL-6 production from days 1–28 p.i. in spleen extracts of R. aurantiacus-infected IL-6+/+ mice and IL-6−/− mice. IL-6 was produced during infection with maximal production on day 3 p.i. No IL-6 was detected in spleen extracts of the IL-6−/− mice. Each point represents the mean ± SD for 6–10 mice.

Article Snippet: Briefly, 100 µl of sample and recombinant mouse IL-6 (Pharmingen, San Jose, CA) were, respectively, added to microtitre wells coated with the purified rat anti-mouse IL-6 mAb (MP5-20F3, Pharmingen), and IL-6 was detected with a biotinylated rat anti-mouse IL-6 mAb (MP5-32C11, Pharmingen).

Techniques: Infection, Produced

Journal:

Article Title: Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

doi: 10.1111/j.1365-2567.2003.01762.x

Figure Lengend Snippet: Histological appearance of livers of IL-6+/+ mice (a), IL-6−/− mice (b), IL-6−/− mice treated with anti-IFN-γ mAb (c), anti-TNF-α mAb (d), and rIL-6 (e) at 2 weeks p.i. A non-necrotic and epithelioid granuloma is seen in the liver of an IL-6+/+ mouse (a), while a large granulomatous lesion with a central area of necrosis is developing in the tissue of IL-6−/− mouse (b). Treatment of IL-6−/− mice with either anti-IFN-γ mAb (c) or anti-TNF-α mAb (d) caused extensive decreases in both number and size of granulomas, and no necrotic granulomas are observed. Administration of rIL-6 reduced the area of necrotic granulomas in the IL-6−/− mouse (e). (original magnification, a, ×400; b–e, ×100).

Article Snippet: Briefly, 100 µl of sample and recombinant mouse IL-6 (Pharmingen, San Jose, CA) were, respectively, added to microtitre wells coated with the purified rat anti-mouse IL-6 mAb (MP5-20F3, Pharmingen), and IL-6 was detected with a biotinylated rat anti-mouse IL-6 mAb (MP5-32C11, Pharmingen).

Techniques:

Journal:

Article Title: Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

doi: 10.1111/j.1365-2567.2003.01762.x

Figure Lengend Snippet: Granuloma formation in the liver at 2 weeks p.i. The mean area of granulomas per field was determined in 10 optical fields of each section from livers of mice treated in the following ways: IL-6+/+ mice; IL-6−/− mice; IL-6−/− mice treated with anti-IFN-γ mAb; IL-6−/− mice treated with anti-TNF-α mAb; and IL-6−/− mice administered rIL-6.

Article Snippet: Briefly, 100 µl of sample and recombinant mouse IL-6 (Pharmingen, San Jose, CA) were, respectively, added to microtitre wells coated with the purified rat anti-mouse IL-6 mAb (MP5-20F3, Pharmingen), and IL-6 was detected with a biotinylated rat anti-mouse IL-6 mAb (MP5-32C11, Pharmingen).

Techniques:

Journal:

Article Title: Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

doi: 10.1111/j.1365-2567.2003.01762.x

Figure Lengend Snippet: Kinetics of bacterial number (Log CFU) of R. aurantiacus-infected IL-6+/+ mice, IL-6−/− mice, IL-6−/− mice treated with anti-IFN-γ mAb, and IL-6−/− mice treated with anti-TNF-α mAb. Four groups of mice were infected with 1 × 108 CFU of R. aurantiacus, and the numbers of viable bacteria present in spleens (a) and livers (b) were determined from days 1–14 p.i. Each point represents the mean ± SD of the R. aurantiacus CFU from 10 mice per time point.

Article Snippet: Briefly, 100 µl of sample and recombinant mouse IL-6 (Pharmingen, San Jose, CA) were, respectively, added to microtitre wells coated with the purified rat anti-mouse IL-6 mAb (MP5-20F3, Pharmingen), and IL-6 was detected with a biotinylated rat anti-mouse IL-6 mAb (MP5-32C11, Pharmingen).

Techniques: Infection

Journal:

Article Title: Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

doi: 10.1111/j.1365-2567.2003.01762.x

Figure Lengend Snippet: Endogenous IFN-γ production in spleen extracts (a) and liver extracts (b) of various mouse groups: infected IL-6+/+ mice, infected IL-6−/− mice, infected IL-6−/− mice treated prophylactically with anti-TNF-α mAb and infected IL-6−/− mice treated prophylactically with rIL-6. Compared with IL-6+/+ mice, a significantly higher level of IFN-γ is observed in the early phase of infection in IL-6−/− mice, whereas IFN-γ production shows a greater decrease at 2 weeks p.i. in IL-6−/− mice. Treatment with anti-TNF-α mAb prevented IFN-γ production. In the IL-6−/− mice treated with rIL-6, the production of IFN-γ in the early phase is observed to decrease. The data are the mean ± SD of 6 mice per group *P < 0·01.

Article Snippet: Briefly, 100 µl of sample and recombinant mouse IL-6 (Pharmingen, San Jose, CA) were, respectively, added to microtitre wells coated with the purified rat anti-mouse IL-6 mAb (MP5-20F3, Pharmingen), and IL-6 was detected with a biotinylated rat anti-mouse IL-6 mAb (MP5-32C11, Pharmingen).

Techniques: Infection

Journal:

Article Title: Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

doi: 10.1111/j.1365-2567.2003.01762.x

Figure Lengend Snippet: Endogenous TNF-α production in liver extracts of various mouse groups: infected IL-6+/+ mice, infected IL-6−/− mice, infected IL-6−/− mice treated prophylactically with anti-IFN-γ mAb and infected IL-6−/− mice treated prophylactically with rIL-6. Compared with IL-6+/+ mice, a significantly higher level of TNF-α is observed in the early phase of infection in IL-6−/− mice. Treatment with either anti-IFN-γ mAb or rIL-6 prevented TNF-α production. The data are the mean ± SD of 6 mice per group *P < 0·01.

Article Snippet: Briefly, 100 µl of sample and recombinant mouse IL-6 (Pharmingen, San Jose, CA) were, respectively, added to microtitre wells coated with the purified rat anti-mouse IL-6 mAb (MP5-20F3, Pharmingen), and IL-6 was detected with a biotinylated rat anti-mouse IL-6 mAb (MP5-32C11, Pharmingen).

Techniques: Infection

Journal: Cancer Research

Article Title: Slit2 Inhibits Breast Cancer Metastasis by Activating M1-Like Phagocytic and Antifibrotic Macrophages

doi: 10.1158/0008-5472.can-20-3909

Figure Lengend Snippet: Figure 5. Slit2 reduces the expression of IL6, thereby inhibiting TAMs activity. A, Total RNA of CD11bþ cells sorted from rSlit2-N PBS-treated MMTV-PyMT tumorswas subjected to gene expression analysis using NanoString technology. The heatmap shows differentially expressed top genes. B, Total RNA was isolated from MDA-MB-231 cells overexpressing Slit2 (231-Slit2) or vector control (231-vec) and analyzed for gene expression using microarray technology. The heatmap shows differentially expressed top genes. C, 231-Slit2 or 231-Vec cells CM was subjected to cytokine array analysis. The representative image shows differentially expressed molecules. D, The graph shows levels of IL6 in CM derived from 231-Sli2 or Vec CM detected by ELISA. E, Expression of Robo1 in MDA-MB-231 cells transduced with lentivirus expressing siRNA specific to Robo1 (si-Robo1) or control (si-ctrl) by Western blot. F, b-actin was used as loading control. MDA-MB-231 parental control, si-Robo1, or si-ctrl from E was treated with rSlit2-N or PBS and phosphorylation at p65 of NFkB (p-NFkB) or total NFkB (t-NFkB) was analyzed. G, Graph showing levels of IL6 in si-Robo1 or si-ctrl MDA-MB-231 cells treated with rSlit2-N or PBS detected by ELISA (n ¼ 3 each group). H, BMDMs were pretreated with serum-free CM (CTRL), or 231-Slit2 CM or 231-Vec CM or 231-Vec CM preincubated with IL6 nAb (231-Vec þ IL6nAb) or IL6 nAb. After 2 hours, pH-Rhodo–labeled S. Aureus particles were added to the cells and recombinant IL6 was added to the 231-Slit2 CM cells (231-Slit2-rIL6). The kinetics of S. Aureus phagocytosis was analyzed using a fluorescence plate reader at every 15 minutes up to 4 hours. , P < 0.05; , P < 0.01; , P < 0.001 using Student t test.

Article Snippet: The cells were pretreated with mouse rSlit2-N (100 ng/mL) or IL6 neutralization antibody (BioXCell# BE0046) for 2 hours.

Techniques: Expressing, Activity Assay, Gene Expression, Isolation, Plasmid Preparation, Control, Microarray, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, Phospho-proteomics, Labeling, Recombinant