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MIF is a critical mediator of the host defense, and is involved in both acute and chronic responses in the lung. Neutralization of MIF reduces neutrophil accumulation into the lung in animal models. We hypothesized that MIF, in the alveolar space, promotes neutrophil accumulation via activation of the CD74 receptor on macrophages.
To determine whether macrophage CD74 surface expression contributes MIF-induced neutrophil accumulation, we instilled recombinant MIF (r-MIF) into the trachea of mice in the presence or absence of anti-CD74 antibody or the MIF specific inhibitor, ISO-1. Using macrophage culture, we examined the downstream pathways of MIF-induced activation that lead to neutrophil accumulation.
Intratracheal instillation of r-MIF increased the number of neutrophils as well as the concentration of macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived chemokine (KC) in BAL fluids. CD74 was found to be expressed on the surface of alveolar macrophages, and MIF-induced MIP-2 accumulation was dependent on p44/p42 MAPK in macrophages. Anti-CD74 antibody inhibited MIF-induced p44/p42 MAPK phosphorylation and MIP-2 release by macrophages. Furthermore, we show that anti-CD74 antibody inhibits MIF-induced alveolar accumulation of MIP-2 (control IgG vs. CD74 Ab; 477.1 ± 136.7 vs. 242.2 ± 102.2 pg/ml, p < 0.05), KC (1796.2 ± 436.1 vs. 1138.2 ± 310.2 pg/ml, p < 0.05) and neutrophils (total number of neutrophils, 3.33 ± 0.93 × 104 vs. 1.90 ± 0.61 × 104, p < 0.05) in our mouse model.
MIF-induced neutrophil accumulation in the alveolar space results from interaction with CD74 expressed on the surface of alveolar macrophage cells. This interaction induces p44/p42 MAPK activation and chemokine release. The data suggest that MIF and its receptor, CD74, may be useful targets to reduce neutrophilic lung inflammation, and acute lung injury.
Macrophage migration inhibitory factor (MIF) is an inflammatory mediator of innate and adaptive immune responses. MIF protein is present in most cells including pituitary cells, T cells, macrophages/monocytes, and is released in response to infection and stress [1]. Plasma MIF concentrations are elevated in patients with inflammatory diseases such as sepsis [2], ARDS [3,4] or rheumatoid arthritis [5]. In addition, plasma concentration of MIF is positively correlated with the severity of sepsis [6,7]. Furthermore, mice deficient in the MIF gene, or those in which the MIF protein has been neutralized, are protected from lethal endotoxemia and septic shock [8,9].
In lung inflammation and injury models, MIP-2 and KC play key roles in neutrophil accumulation into the lungs [10]. Neutrophils are an important component of the inflammatory response in acute lung injury [11,12]. The elimination of neutrophils can markedly decrease the severity of lung injury in animal lung inflammation models [13]. MIF levels are increased in BAL fluid in lipopolysaccharide (LPS)-induced lung injury model [14], and MIF neutralizing antibody blocks LPS-induced pulmonary neutrophil accumulation in animal models [15,16], suggesting that MIF can influence neutrophil accumulation into the lungs.
CD74 (also known as a MHC class II invariant chain) is a type II transmembrane protein, reported to be part of the MIF receptor complex [17]. Several studies have shown that CD74 is expressed both intracellularly and on the cell surface in B cell lymphoma [18], T cell lymphoma, melanoma cells [19] and gastric epithelial cells [20]. MIF binds to cell surface CD74, and induces p44/p42 MAPK phosphorylation and cell proliferation. Moreover neutralization of CD74 inhibits MIF-induced cell proliferation in B cells and fibroblasts [17]. Other recent studies have shown that anti-CD74 antibody blocks MIF-CD74 binding on the cell surface of gastric epithelial cells [21], and anti-CD74 antibody attenuated proliferation of prostate cancer cells [22]. CD74 has a short N-terminal cytoplasmic domain of 28 amino acids and appears to lack intracellular signaling domains. Recently, CD44 has been identified as an accessory protein required for MIF-CD74 signal transduction [23].
MIF is found at increased levels in BAL fluids from both LPS induced lung inflammation [14] and polymicrobial sepsis models [24]. BAL fluid-MIF levels in ARDS patients were also significantly increased compared with healthy controls [3]. However, little is known about the mechanisms involved in MIF-induced lung inflammation. We have previously shown that MIF itself causes neutrophil accumulation into the alveolar space [25]. MIF is an intracellular protein that can be released into the extracellular environment where it acts as a potent inflammatory stimulant. Extracellular MIF can bind to the cell surface molecule CD74.
Therefore we focused on the MIF receptor CD74 in an animal model. We hypothesized that MIF, in the alveolar space, results in neutrophil accumulation via activation of the CD74. Here we used intra-tracheal instillation of MIF, and studied the contribution of CD74 in MIF-induced neutrophil accumulation in a mouse model.
The present study demonstrated that MIF instillation increased the concentration of MIP-2 and KC as well as the number of neutrophils in the alveolar space. This study shows that CD74 expressed on the cell surface of alveolar macrophages, contributes to the MIF-induced neutrophil accumulation into the alveolar space.
Mouse model
All experiments were approved by the Institutional Animal Care and Use Committee at The Feinstein Institute for Medical Research at North Shore-Long Island Jewish Health System. C57Bl/6 male mice (8–12 weeks old) were anesthetized with isoflurane and the trachea was surgically exposed. Recombinant murine MIF (r-MIF; 1.0 μg in 50 μl normal saline) or 50 μl normal saline alone (control) was instilled directly into the lungs via the trachea using a 29 gauge needle. Groups of mice (n = 6/group) were euthanized at 3, 6 and 20 hours post instillation, blood was collected by cardiac puncture. The blood samples were centrifuged (2000 × g, 10 min), and the plasma was stored at -80°C for further examination. Postmortem bronchoalveolar lavage (BAL) was performed by instilling and withdrawing sterile physiological saline (1 ml) through a tracheal cannula using a 20 gauge Surflo i.v. catheter (Terumo, Elkton, MD). This procedure was repeated three times, and the three BAL fluid samples were pooled. Lung tissues were then isolated and frozen immediately in liquid nitrogen. The BAL fluid was centrifuged (300 × g, 5 min), and the supernatant portions were stored at -80°C for further examination.
Differential cell count in BAL fluid
Immediately after collection of BAL fluids, erythrocytes were lysed using 0.2% saline and the remaining cells were resuspended in Hanks' Balanced Salt Solution (Invitrogen, Grand Island, NY). Total cell count of each BAL sample was determined using a Neubauer hemocytometer (Hausser Scientific, Horsham, PA). Differential cell counts were performed (200 cells for each experimental condition) on cytospin slides stained with Protocol HEMA3 solution (Fisher scientific, Fair Lawn, NJ). Total protein concentration in BAL fluid was measured using Coomassie protein assay kit (Thermo Scientific, Rockford, IL).
Reagents and antibodies
r-MIF was prepared from an Escherichia coli expression system, and treated with polymyxin B as previously described [26]. The MIF specific inhibitor (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) was diluted for use with a minimal amount of dimethyl sulfoxide (DMSO, Fisher Scientific, Fair Lawn, NJ) as previously described [24,27,28]. Anti-CD74 antibody (clone; In-1) for western blotting was purchased from BD Bioscience (San Jose, CA), and anti-CD74 goat polyclonal antibody for flow cytometry and immunofluorescence was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-phospho p44/p42 MAPK, anti-phospho p38 MAPK, anti-p38 MAPK, anti-phospho JNK, anti-JNK and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). Stripping buffer for membrane was purchased from Thermo Scientific (Rockford, IL). Anti-p44/p42 MAPK antibody and isotype control goat IgG were purchased from Santa Cruz Biotechnology Inc. MAPK specific inhibitor PD98059 and p38 MAPK specific inhibitor SB202190 were purchased from Calbiochem (San Diego, CA).
Cell culture
Murine macrophages (RAW264.7) and murine alveolar type II epithelial cells (MLE-12) were purchased from American Type Culture Collection (Manassas, VA). RAW264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), L-glutamine, penicillin and streptomycin (Invitrogen, Grand Island, NY) at 37°C in a 5% CO2 humidified incubator. MLE-12 cells were cultured in DMEM/F12 media supplemented with 2% FCS, 15 mM HEPES, L-glutamine, penicillin and streptomycin [29,30]. In experiments of anti-CD74 antibody and ISO-1 treatment in vitro, RAW264.7 cells were treated with 10 μg/ml anti-CD74 antibody or 10 μg/ml control goat IgG at 37°C for 30 min. After 30 min pre-treatment, cells were stimulated with 100 ng/ml MIF at 37°C for 10 min. In addition, 100 mM ISO-1 in PBS or 5% DMSO in PBS and 100 ng/ml MIF were mixed in microtubes at 37°C for 30 min, then cells were stimulated with the mixture at 37°C for 10 min. Cells were lysed and subjected to SDS-PAGE, then immunoprobed by anti-phospho and total p44/p42 MAPK antibody.
Western blot analysis
A total of 1.0 × 106 RAW264.7 cells/sample were stimulated with various concentrations of r-MIF in 1% FCS containing media. After washing (×2) with ice cold PBS buffer, cells were lysed in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM EDTA (pH 8.0), 1% octylphenol polyethyleneglycol (NP-40), 0.5% deoxycholic acid, 0.1% SDS, and 1 mM PMSF. Lysates were separated from debris by centrifugation (7800 × g) for 15 min, and lysates were boiled for 5 min in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) under reducing conditions. Tissues were thawed and homogenized in lysis buffer at 4°C, and incubated on ice for 30 min, then centrifuged (7800 × g) for 15 min at 4°C. The supernatants were analyzed for protein content, and were boiled for 5 min in Laemmli sample buffer (Bio-Rad Laboratories). Proteins (total protein content was 15 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidine difluoride membrane (Millipore, Billerica, MA). Prestained molecular weight standards (Crystalgen Inc., Plainview, NY) were run with each gel to determine the approximate molecular weight of detected bands. Then membranes were incubated with the specific primary antibodies and horse radish peroxidase (HRP) conjugated secondary antibodies. After washing with tris-buffered saline contained 0.1% tween-20 (TBS-T), membranes were incubated with Chemiluminescence Luminol Reagent (Amersham Biosciences, Pittsburgh, PA) and exposed to photographic film (Fujifilm Corp, Tokyo, Japan). Protein bands were quantified by densitometric analysis using a Gel Doc 2000 Chemi Doc scanner (Bio-Rad Laboratories) and Quantity One 4.4.0 software.
Cell staining and flow cytometry
Single-cell suspensions were incubated at 4°C for 2 hours with anti-CD74 antibody or control IgG diluted in staining buffer (PBS containing 2% FCS). Cells were washed with staining buffer, and then incubated at 4°C for 30 min with FITC-conjugated secondary antibody. Flow cytometry analysis was performed on FACSCalibur (BD Biosciences, San Jose, CA) and data were analyzed using CellQuest software.
Cytokine analysis
Cytokine concentrations were evaluated using commercially available enzyme-linked immunosorbent assay (ELISA) kits for keratinocyte-derived chemokine (KC; CXCL1) and macrophage inflammatory protein 2 (MIP-2; CXCL2) from R&D Systems. (Minneapolis, MN).
Histological study
To obtain lungs for routine histology, the trachea was cannulated and the lungs were gently fixed at inflation with 4% paraformaldehyde solution. The fixed lungs were sectioned (5 μm), and the sections stained with hemotoxylin and eosin (Sigma-Aldrich, St. Louis, MO). For identification of neutrophils, the sections were stained with naphthol AS-D chloroacetate esterase (Sigma-Aldrich, St. Louis, MO).
Immunofluorescence study
Cells from BAL fluid were prepared using cytospin centrifugation. The slides were fixed in 4% paraformaldehyde in PBS, pH 7.4 for 10 min at room temperature (RT) and permeabilized with 0.01% Triton X-100 in PBS for 5 min at RT. After washing with PBS, each slide was blocked with 10% rabbit serum in PBS for 1 hour at RT. Slides were then incubated with anti-CD74 polyclonal antibody or control IgG diluted in blocking solution for 18 hours at 4°C, and were incubated with FITC-conjugated secondary antibody diluted in blocking solution for 1 hour at RT. After washing with PBS, slides were incubated with 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI, AnaSpec Inc. San Jose, CA) in blocking solution for 30 min at RT. Cover slips were then placed on the slides with mounting medium (Vector laboratories, Inc. Burlingame, CA). Images were obtained using a Zeiss ApoTome fluorescent microscope and Zeiss Axiovision software (Zeiss LSM, Thornwood, NY).
Statistical analysis
All values are expressed as mean ± standard deviation (SD). Multiple groups were compared using analysis of variance (ANOVA) using the Dunnett's post hoc test. Results were considered statistically significant at p < 0.05.
MIF induces neutrophil accumulation into the lung
We have previously reported that MIF has the ability to induce neutrophil accumulation into the alveolar space of mice [25]. To examine the kinetics of MIF-induced leukocytosis, C57Bl/6 mice were instilled intratracheally with r-MIF and the influx of neutrophils was examined over time. The number of neutrophils in BAL fluid at 3, 6 or 20 hours after instillation of MIF significantly increased compared to control animals in which normal saline was instilled alone (Fig. 1A). The number of neutrophils was highest at 6 hours post instillation (total number of neutrophils, saline; 0.09 ± 0.04 × 104, 3 hours; 2.95 ± 1.13 × 104, 6 hours; 5.10 ± 1.35 × 104, 20 hours; 4.18 ± 1.31 × 104). At 6 hours post instillation neutrophil number increased not only in the BAL fluid (Fig. 1B, i and 1B, iv) but also in the alveolar tissue (Fig. 1B, ii, iii, v, vi). BAL fluids were assessed for total protein content, and the neutrophil chemoattractants MIP-2 and KC. Total protein concentration in BAL fluids significantly increased at 6 hours after MIF instillation (Fig. 2A, saline; 71.9 ± 21.1 μg/ml, 3 hours; 130.4 ± 58.5 μg/ml, 6 hours; 158.7 ± 53.6 μg/ml, 20 hours; 127.9 ± 44.5 μg/ml). Furthermore, MIP-2 and KC in BAL fluids increased at 3, 6 hours after instillation and peaked around 3 hours (Fig. 2B, the concentration of MIP-2 at 3 hours; 422.7 ± 125.7 pg/ml, Fig. 2C, the concentration of KC at 3 hours; 1859.4 ± 684.1 pg/ml). However, MIP-2 and KC concentrations in plasma from these mice did not significantly change following MIF instillation (data not shown).
Figure 1. MIF induces neutrophil accumulation into the alveolar space. Mice were euthanized at 3, 6 or 20 hours post instillation of 1.0 μg MIF or 3 hours (saline controls). Lungs were lavaged three times with normal saline. A. The number of neutrophil in the bronchoalveolar lavage (BAL) fluid was counted by hemocytometer. Bars indicate mean ± SD (n = 6/group). *P < 0.05 compared with normal saline control. B. (i), (iv) show the cell population in BAL fluid with HEMA3 stain. (i)-(iii) shows representative samples from a control mouse, and (iv)-(vi) shows samples from 6 hours after MIF instilled mice. Lung tissue stained with H-E stain in (ii), (v) and naphthol AS-D chloroacetate esterase stain (neutrophil granules were stained with bright red color, point with arrows) in (iii), (vi).
Figure 2. Alveolar protein and chemokine response following intratracheal instillation of MIF. Mice were euthanized at 3, 6 or 20 hours post instillation of MIF, and the lungs were lavaged with normal saline. A. Total protein concentration in BAL fluid after 3, 6, 20 hours. B. MIP-2 concentration in BAL fluid after 3, 6, 20 hours measured by ELISA. C. KC concentration in BAL fluid measured by ELISA. Bars indicate mean ± SD (n = 6/group). *P < 0.05 compared with normal saline control.
Macrophages express MIF receptor component CD74
MIF intratracheal instillation markedly induced neutrophil accumulation in the lung. To determine the involvement of CD74 in the MIF-induced responses, we examined CD74 expression in several cell types. We used RAW264.7 cells and the MLE-12 cells as models of alveolar macrophages and type II alveolar epithelial cells respectively, and assayed for the presence of CD74 in RAW264.7, MLE-12 and lung tissues. Two isoforms of CD74 (p41 and p31) were observed. There was higher expression of CD74 protein in RAW264.7 and lung tissues than in MLE-12 cells (Fig. 3A). To confirm CD74 expression in primary alveolar macrophages in BAL fluid, we used immunofluorescence microscopy. We found that CD74 protein is also expressed in alveolar macrophages (Fig. 3B). More than 95% of the cells in BAL fluid were recognized as macrophages using stained cytospin slides in this experiment. Furthermore, cell surface expression of CD74 was evident in non-permeabilized RAW264.7 and alveolar macrophages but not in MLE-12 (Fig. 3C).
Figure 3. CD74 expresses on the cell surface of alveolar macrophages. A. RAW264.7, MLE-12 and lung tissues from two non-treated mice were homogenized in lysis buffer and prepared for western blot analysis for CD74. The blots were then stripped and re-probed for GAPDH antibody as a loading control (total protein content was 15 μg/lane). Data represents three independent experiments. B. BAL cells from mice were stained with anti-CD74 antibody or control IgG and subsequent FITC-conjugated secondary antibody after fixation and permeabilization. (i), (ii) show the control IgG and (iii), (iv) show anti-CD74 antibody. (i), (iii) show differential interference contrast (DIC) and (ii), (iv) show fluorescent image. Green color represents CD74 or control IgG (FITC) and blue color represents nuclear (DAPI). Scale bar represents 20 μm. Data represents four independent experiments. C. Flow cytometry of non-permeabilized RAW264.7, MLE-12 and alveolar macrophages assessing surface expression of CD74. Shaded histograms show CD74 staining, and opened histograms show the control IgG staining. Data shown is representative of three independent experiments.
MIF activates p44/p42 MAPK pathway and stimulates MIP-2 release from macrophages
Following MIF stimulation, the p44/p42 MAPK signaling pathway was activated in both a time and dose dependent manner in RAW264.7 macrophage cells (Fig. 4A, 4B). However, there was no significant activation of p38 MAPK or JNK signaling pathways (data not shown). To investigate whether MIF stimulation induces the release of neutrophil chemokines, we measured MIP-2 and KC in cell culture supernatants. There was a significant accumulation of MIP-2 (Fig. 4C, MIP-2 at 100 ng/ml stimulation; 1243.1 ± 35.3 pg/ml) but not KC (data not shown) in the culture media following stimulation with MIF. To confirm that activation of MIP-2 is downstream of p44/p42 MAPK following MIF stimulation, specific MAP kinase inhibitors were used. The p44/p42 MAPK specific inhibitor PD98059 attenuated the MIF-induced MIP-2 accumulation (Fig. 5, MIF; 1219.7 ± 48.1 pg/ml, MIF + PD; 752.2 ± 42.6 pg/ml, p < 0.05). However, the p38 MAPK inhibitor SB202190 had no effect on the MIP-2 accumulation (Fig. 5, MIF + SB; 1307.6 ± 68.8 pg/ml). This suggests that MIF-induced MIP-2 accumulation depends, at least in part, on p44/p42 MAPK signaling pathway.
Figure 4. MIF activates p44/p42 MAPK pathway and stimulates MIP-2 release from macrophages. A. RAW264.7 cells were stimulated with 100 ng/ml MIF for 5, 10, 15, 30 or 60 min. After stimulation the cells were lysed and boiled for SDS-PAGE analysis. Transferred proteins were detected by anti-phospho and total p44/p42 MAPK antibodies. Data represents four independent experiments. B. RAW264.7 cells were stimulated with 5, 20, 100 ng/ml MIF for 10 min. Proteins were detected by anti-phospho and total p44/p42 MAPK antibodies. Data represents three independent experiments. C. RAW264.7 cells were stimulated with different concentrations of MIF (20, 50, 100 ng/ml) or control media for 18 hours at 37°C. After 18 hours supernatants were collected and MIP-2 concentrations were measured by ELISA. Bars indicate mean ± SD (n = 3/group). Data is representative of four independent experiments. *P < 0.05 compared with control media.
Figure 5. MIF-induced MIP-2 accumulation depends on p44/p42 MAPK pathway. RAW264.7 cell were incubated with MAP kinase kinase (MEK) specific inhibitor (PD98059, 30 mM) or p38 MAP kinase specific inhibitor (SB202190, 30 mM) or DMSO for 30 min before stimulation with 100 ng/ml MIF. After 18 hours stimulation, supernatants were collected and MIP-2 concentrations in supernatants were measured by ELISA. Bars indicate mean ± SD (n = 3/group). Data is representative of three independent experiments. *P < 0.05 compared with DMSO + MIF.
Anti-CD74 antibody inhibits MIF-induced p44/p42 MAPK phosphorylation and MIP-2 accumulation in macrophages
MIF signaling occurs following MIF binding to CD74. To examine whether neutralization of CD74 can inhibit the MIF-induced cell signaling, we used a CD74 antibody, and the specific MIF inhibitor, ISO-1 [24,27,28]. We found that both CD74 antibody and ISO-1 treatment significantly inhibited MIF-induced phosphorylation of p44/p42 MAPK (Fig. 6A, relative ratio, MIF + control IgG; 2.98 ± 0.33, MIF + CD74 Ab; 2.07 ± 0.19, p < 0.05, MIF + DMSO; 2.96 ± 0.38, MIF + ISO-1; 1.79 ± 0.27, p < 0.05). Furthermore, both ISO-1 and CD74 antibody treatment inhibited MIF-induced MIP-2 accumulation in culture media (Fig. 6B, MIF + control IgG; 1348.7 ± 107.3 pg/ml, MIF + CD74 Ab; 873.0 ± 47.1 pg/ml, p < 0.05, MIF + DMSO; 1372.2 ± 34.6 pg/ml, MIF + ISO-1; 635.7 ± 42.5 pg/ml, p < 0.05). These data suggest that anti-CD74 antibody has a similar effect as ISO-1 treatment in inhibiting MIF-induced cell activation.
Figure 6. Anti-CD74 antibody treatment inhibits MIF-induced p44/p42 MAPK phosphorylation and MIP-2 accumulation in macrophages. A. RAW264.7 cells were treated with 10 μg/ml anti-CD74 antibody or control IgG, or 100 mM ISO-1 or DMSO control for 10 min. Cells were lysed and subjected to SDS-PAGE, immunoprobed by anti-phospho and total p44/p42 MAPK antibody. Bar graphs show the density ratio of phospho- vs. total-p44/p42 MAPK bands. Bar graphs represent the mean ± SD of three independent experiments. B. RAW264.7 cells were treated with 10 μg/ml anti-CD74 antibody or control IgG, or 100 mM ISO-1 or DMSO control for 18 hours. After 18 hours supernatant were collected and MIP-2 concentration were measured by ELISA. Bars indicate mean ± SD (n = 3/group). Data represents four independent experiments. *P < 0.05 compared with control IgG + MIF or DMSO + MIF stimulation.
Anti-CD74 antibody inhibits MIF-induced neutrophil accumulation into the alveolar space
In the next series of experiments, MIF was intratracheally instilled in the presence of anti-CD74 antibody or ISO-1, an MIF inhibitor that blocks binding to CD74. Mixtures of either 1.0 μg MIF and 10 μg anti-CD74 antibody or 1.0 μg MIF and 0.5 μg ISO-1 were instilled via the trachea to ensure identical distribution of both agonist and inhibitor. Anti-CD74 antibody and ISO-1 significantly inhibited MIF-induced neutrophil accumulation into the lung (Fig. 7A, total number of neutrophils, MIF + control IgG; 3.33 ± 0.92 × 104, MIF + CD74 Ab; 1.90 ± 0.61 × 104, p < 0.05, MIF + DMSO; 3.65 ± 0.82 × 104, MIF + ISO-1; 1.53 ± 0.69 × 104, p < 0.05). To assess the effect of anti-CD74 antibody treatment on chemokine accumulation, MIP-2 and KC concentrations were measured in the BAL fluids. Anti-CD74 antibody and ISO-1 treatment significantly inhibited the MIF-induced MIP-2 (Fig. 7B, MIF + control IgG; 477.1 ± 136.7 pg/ml, MIF + CD74 Ab; 242.2 ± 102.2 pg/ml, p < 0.05, MIF + DMSO; 405.3 ± 130.5 pg/ml, MIF + ISO-1; 210.6 ± 60.6 pg/ml, p < 0.05) and KC accumulation in BAL fluids (Fig. 7C, MIF + control IgG; 1796.2 ± 436.1 pg/ml, MIF + CD74 Ab; 1138.2 ± 310.3 pg/ml, p < 0.05, MIF + DMSO; 1659.8 ± 444.5 pg/ml, MIF + ISO-1; 763.4 ± 271.4 pg/ml, p < 0.05). Taken together, anti-CD74 antibody and ISO-1 both had an inhibitory effect on MIF-induced MIP-2, KC accumulation and resultant neutrophil accumulation into the alveolar space. These data suggest that CD74 has a pivotal role in MIF-induced neutrophil accumulation into the alveolar space.
Figure 7. Anti-CD74 antibody treatment inhibits MIF-induced neutrophil accumulation into the alveolar space. A. The number of neutrophils in BALF from treated mice. The groups of mice were instilled via the trachea with either 1.0 μg MIF plus 10 μg anti-CD74 antibody or 1.0 μg MIF plus 10 μg control IgG. The other groups of mice were instilled with either 1.0 μg MIF plus 0.5 mg ISO-1 or 1.0 μg MIF plus 5% DMSO. Mice were euthanized 3 hours post instillation, then lungs were lavaged three times with normal saline. The number of neutrophils were counted using a hemocytometer. *P < 0.05 compared with control IgG + MIF or DMSO + MIF stimulation, B. MIP-2, and C. KC concentration in BAL fluid from 3 hours post instilled mice measured by ELISA. Bars indicate mean ± SD (n = 6/group). *P < 0.05 compared with control IgG + MIF or DMSO + MIF stimulation.
MIF is expressed in multiple immune and nonimmune cell types and is released in response to infection and other stresses [1]. MIF exists as a homotrimer, each monomer being approximately 12.5 kDa. MIF has enzymatic activities, and is a potent regulator of innate and adaptive immune responses [1]. MIF has immunoregulatory functions in sepsis [2], ARDS[3], bronchial asthma [31], rheumatoid arthritis [5] and tumorgenesis [32].
Neutrophils play an important role in the inflammatory response, and can be associated with severe lung injury in patients with the acute respiratory distress syndrome (ARDS) [11]. Previous studies suggest that MIF participates in neutrophil accumulation into the lung after intraperitoneal LPS injection [15]. In the LPS intratracheal instillation model, neutralization of MIF attenuated capillary leak and the levels of TNF-α and IL-6 in BAL fluid [14]. The elimination of neutrophils, using anti-neutrophil antibody, markedly decreases the severity of animal acute lung injury in animal models [13]. Neutrophil recruitment from blood into tissue at sites of inflammation usually occurs in post-capillary venules and requires capture, rolling and adhesion on endothelial cells in acute lung injury [12]. A multitude of molecules including selectin, integrin, and immunoglobulin adhesion molecules, cytokines and chemokines participate in this sequential process in a variety of vascular beds [33,34]. The CXC chemokine interleukin-8 (IL-8) has been implicated in mediating the influx of neutrophils into the lung in ARDS patients [35,36], particularly sepsis-associated ARDS [37]. The murine equivalents of IL-8, MIP-2 and KC, have been reported to be the two most crucial chemokines for neutrophil recruitment [38]. Neutralization of MIP-2 significantly decreases neutrophil recruitment into the lung [39]. Both MIP-2 and KC bind to CXCR2 receptors, and blockade of CXCR2 attenuates neutrophil influx into the lung [40-43].
In the present study, we investigated the contribution of macrophage CD74 in MIF-induced neutrophil accumulation into the alveolar space. We showed previously that MIF has the ability to induce neutrophil accumulation [25]. These data suggest that MIF stimulates some types of cells in the alveolar space, to release MIP-2 and KC, which result in neutrophil accumulation in the alveolar space. Although the number of neutrophils in the alveolar space was less than in bacteria or bacterial component induced lung injury models, the current study showed that MIF administration can result in neutrophil accumulation (Fig. 1). While MIF has potent inflammatory and immunoregulatory properties, the mechanisms of cellular binding and activation remain to be fully elucidated. The extracellular domain of CD74 has been shown to bind MIF with high affinity and act as a surface membrane receptor [17]. Theoretically, MIF signaling is initiated after MIF binding to cell surface CD74. Recently, others have reported that expression of CD74 in mouse lung sections was predominantly localized to alveolar macrophage and type II alveolar epithelial cells [44]. Therefore we examined CD74 protein level and CD74 cell surface expression in macrophages and epithelial cells. Interestingly, we found that CD74 is expressed on the alveolar macrophage cell surface, suggesting that macrophages could respond to extracellular MIF (Fig. 3). In addition, we have shown that macrophages can release MIP-2 following MIF stimulation (Fig. 4C), and MIF-induced MIP-2 accumulation depends, at least in part, on p44/p42 MAPK signaling pathways as indicated in the specific inhibitor study in macrophages (Fig. 5).
The neutralization of MIF is a promising approach to develop new anti-inflammatory agents and treatments for diseases that involve increased MIF production or release [45,46]. In fact, an administration of neutralizing anti-MIF antibodies has proven therapeutically effective in animal models of sepsis [9] and in LPS-induced lung injury models [15,16]. X-ray crystallography studies show that ISO-1, an MIF-specific inhibitor, binds to the inflammatory active site of MIF in [27,28]. Treatment with ISO-1 has been reported to be effective in endotoxemia [27], polymicrobial sepsis [24] and autoimmune diabetes models [47]. Leng and colleagues have shown that anti-CD74 antibody can block MIF-induced cell proliferation in B cells and fibroblast cells [17]. Other recent studies have demonstrated that anti-CD74 antibody blocks MIF-CD74 binding in the cell surface of gastric epithelial cells [21], and anti-CD74 antibody attenuated proliferation of prostate cancer cells [22]. Moreover, a humanized anti-CD74 monoclonal antibody (hLL1) treatment has been reported to have therapeutic potential for B-cell malignancies [48]. In our study, we have shown that anti-CD74 antibody significantly inhibited the MIF-induced p44/p42 MAPK phosphorylation and MIP-2 accumulation (Fig. 6), suggesting that the MIF-induced MIP-2 accumulation depends, at least in part, on cell surface CD74 of macrophages.
We used anti-CD74 antibody in our mouse model, and have shown that anti-CD74 antibody significantly inhibited the MIF-induced neutrophil accumulation into the alveolar space as well as accumulation of MIP-2, KC (Fig. 7). The results revealed that anti-CD74 antibody has the ability to inhibit MIF-induced neutrophil accumulation in vivo. In this study, we have instilled mixtures of either MIF and ISO-1 or MIF and anti-CD74 antibody to ensure that MIF and inhibitor are delivered to an identical segment of the lung. This is important, because we believe that the native MIF binds to the receptor (CD74) to effect the inflammatory response and that the inhibitor or antibody blocks the MIF-receptor interaction. Future studies will address the question of whether the inflammatory response induced by MIF-CD74 interactions can be attenuated by intravenous administration of ISO-1 or anti-CD74 antibody, and the effective time course of administration. We believe a combination of CD74 antibody and other anti-inflammatory agents might be beneficial for treatment of neutrophilic lung inflammation.
Anti-CD74 antibody treatment reduced the number of neutrophils compared to control at 43%. This suggests that anti-CD74 antibody had a partial inhibitory effect in MIF-induced neutrophil accumulation. The data raises the possibility that MIF could activate via other signaling pathways, not CD74 alone. Indeed, an MIF endocytotic pathway has been proposed, in which MIF is taken up by the cell and binds to Jun activation domain-binding protein (Jab1) (COP9 signalosome subunit 5; CNS5). MIF would thereby inhibit Jab1-enhanced AP-1 transcriptional activity [49]. A recent study has shown that MIF activates p44/p42 MAPK pathway rapidly and transiently in a Jab1 dependent manner [50].
This study shows that CD74 is expressed on the cell surface of alveolar macrophages, and potentially contributes to MIF-induced neutrophil accumulation into the alveolar space. We have demonstrated that anti-CD74 antibody inhibits MIF-induced p44/p42 MAPK phosphorylation and MIP-2 release in macrophages. Furthermore, we have demonstrated that anti-CD74 antibody treatment inhibits MIF-induced MIP-2, KC accumulation and neutrophil accumulation in mouse model. The data suggest that MIF and its receptor CD74 may be useful targets to reduce neutrophilic lung inflammation, and acute lung injury.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KT carried out the experimental work, the data analysis and drafted the manuscript. KK, HML, YZ and XL participated in the discussion of the experiments. KO provided MAPK specific inhibitors and edited the manuscript. CNM provided r-MIF and edited the manuscript. YA provided MIF specific inhibitor, ISO-1. EJM provided overall leadership to the experimental design, data analysis, and preparation of the manuscript.
This work was supported by NIH R01 HL 081655 (EJM) and Swedish Research Council K2009-77PK-20982-01-1 (HML).
Calandra T, Roger T: Macrophage migration inhibitory factor: a regulator of innate immunity.
Bozza FA, Gomes RN, Japiassu AM, Soares M, Castro-Faria-Neto HC, Bozza PT, Bozza MT: Macrophage migration inhibitory factor levels correlate with fatal outcome in sepsis.
Donnelly SC, Haslett C, Reid PT, Grant IS, Wallace WA, Metz CN, Bruce LJ, Bucala R: Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome.
Gao L, Flores C, Fan-Ma S, Miller EJ, Moitra J, Moreno L, Wadgaonkar R, Simon B, Brower R, Sevransky J, et al.: Macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations.
Leech M, Metz C, Hall P, Hutchinson P, Gianis K, Smith M, Weedon H, Holdsworth SR, Bucala R, Morand EF: Macrophage migration inhibitory factor in rheumatoid arthritis: evidence of proinflammatory function and regulation by glucocorticoids.
Lehmann LE, Novender U, Schroeder S, Pietsch T, von Spiegel T, Putensen C, Hoeft A, Stuber F: Plasma levels of macrophage migration inhibitory factor are elevated in patients with severe sepsis.
Chuang CC, Wang ST, Chen WC, Chen CC, Hor LI, Chuang AY: Increases in serum macrophage migration inhibitory factor in patients with severe sepsis predict early mortality.
Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, David JR: Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.
Calandra T, Echtenacher B, Roy DL, Pugin J, Metz CN, Hultner L, Heumann D, Mannel D, Bucala R, Glauser MP: Protection from septic shock by neutralization of macrophage migration inhibitory factor.
Gupta S, Feng L, Yoshimura T, Redick J, Fu SM, Rose CE Jr: Intra-alveolar macrophage-inflammatory peptide 2 induces rapid neutrophil localization in the lung.
Abraham E: Neutrophils and acute lung injury.
Reutershan J, Ley K: Bench-to-bedside review: acute respiratory distress syndrome – how neutrophils migrate into the lung.
Abraham E, Carmody A, Shenkar R, Arcaroli J: Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury.
Rittirsch D, Flierl MA, Day DE, Nadeau BA, McGuire SR, Hoesel LM, Ipaktchi K, Zetoune FS, Sarma JV, Leng L, et al.: Acute lung injury induced by lipopolysaccharide is independent of complement activation.
Makita H, Nishimura M, Miyamoto K, Nakano T, Tanino Y, Hirokawa J, Nishihira J, Kawakami Y: Effect of anti-macrophage migration inhibitory factor antibody on lipopolysaccharide-induced pulmonary neutrophil accumulation.
Matsuda N, Nishihira J, Takahashi Y, Kemmotsu O, Hattori Y: Role of macrophage migration inhibitory factor in acute lung injury in mice with acute pancreatitis complicated by endotoxemia.
Leng L, Metz CN, Fang Y, Xu J, Donnelly S, Baugh J, Delohery T, Chen Y, Mitchell RA, Bucala R: MIF signal transduction initiated by binding to CD74.
Wraight CJ, van Endert P, Moller P, Lipp J, Ling NR, MacLennan IC, Koch N, Moldenhauer G: Human major histocompatibility complex class II invariant chain is expressed on the cell surface.
Ong GL, Goldenberg DM, Hansen HJ, Mattes MJ: Cell surface expression and metabolism of major histocompatibility complex class II invariant chain (CD74) by diverse cell lines.
Barrera CA, Beswick EJ, Sierra JC, Bland D, Espejo R, Mifflin R, Adegboyega P, Crowe SE, Ernst PB, Reyes VE: Polarized expression of CD74 by gastric epithelial cells.
Beswick EJ, Pinchuk IV, Suarez G, Sierra JC, Reyes VE: Helicobacter pylori CagA-dependent macrophage migration inhibitory factor produced by gastric epithelial cells binds to CD74 and stimulates procarcinogenic events.
Meyer-Siegler KL, Iczkowski KA, Leng L, Bucala R, Vera PL: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.
Shi X, Leng L, Wang T, Wang W, Du X, Li J, McDonald C, Chen Z, Murphy JW, Lolis E, et al.: CD44 is the signaling component of the macrophage migration inhibitory factor-CD74 receptor complex.
Sakuragi T, Lin X, Metz CN, Ojamaa K, Kohn N, Al-Abed Y, Miller EJ: Lung-derived macrophage migration inhibitory factor in sepsis induces cardio-circulatory depression.
Lin X, Sakuragi T, Metz CN, Ojamaa K, Skopicki HA, Wang P, Al-Abed Y, Miller EJ: Macrophage migration inhibitory factor within the alveolar spaces induces changes in the heart during late experimental sepsis.
Bernhagen J, Mitchell RA, Calandra T, Voelter W, Cerami A, Bucala R: Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF).
Al-Abed Y, Dabideen D, Aljabari B, Valster A, Messmer D, Ochani M, Tanovic M, Ochani K, Bacher M, Nicoletti F, et al.: ISO-1 binding to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in severe sepsis.
Lubetsky JB, Dios A, Han J, Aljabari B, Ruzsicska B, Mitchell R, Lolis E, Al-Abed Y: The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents.
Sharma AK, Fernandez LG, Awad AS, Kron IL, Laubach VE: Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury.
Lee VY, Schroedl C, Brunelle JK, Buccellato LJ, Akinci OI, Kaneto H, Snyder C, Eisenbart J, Budinger GR, Chandel NS: Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway.
Rossi AG, Haslett C, Hirani N, Greening AP, Rahman I, Metz CN, Bucala R, Donnelly SC: Human circulating eosinophils secrete macrophage migration inhibitory factor (MIF). Potential role in asthma.
Bucala R, Donnelly SC: Macrophage migration inhibitory factor: a probable link between inflammation and cancer.
Springer TA: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.
Ley K: Integration of inflammatory signals by rolling neutrophils.
Miller EJ, Cohen AB, Nagao S, Griffith D, Maunder RJ, Martin TR, Weiner-Kronish JP, Sticherling M, Christophers E, Matthay MA: Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality.
Donnelly SC, Strieter RM, Kunkel SL, Walz A, Robertson CR, Carter DC, Grant IS, Pollok AJ, Haslett C: Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups.
Miller EJ, Cohen AB, Matthay MA: Increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis.
Olson TS, Ley K: Chemokines and chemokine receptors in leukocyte trafficking.
Greenberger MJ, Strieter RM, Kunkel SL, Danforth JM, Laichalk LL, McGillicuddy DC, Standiford TJ: Neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia.
Mehrad B, Strieter RM, Moore TA, Tsai WC, Lira SA, Standiford TJ: CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis.
Hayashi S, Yatsunami J, Fukuno Y, Kawashima M, Miller EJ: Antileukinate, a hexapeptide inhibitor of CXC-chemokine receptor, suppresses bleomycin-induced acute lung injury in mice.
Lomas-Neira JL, Chung CS, Grutkoski PS, Miller EJ, Ayala A: CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.
Lin X, Yang H, Sakuragi T, Hu M, Mantell LL, Hayashi S, Al-Abed Y, Tracey KJ, Ulloa L, Miller EJ: Alpha-chemokine receptor blockade reduces high mobility group box 1 protein-induced lung inflammation and injury and improves survival in sepsis.
Marsh LM, Cakarova L, Kwapiszewska G, von Wulffen W, Herold S, Seeger W, Lohmeyer J: Surface expression of CD74 by type II alveolar epithelial cells: a potential mechanism for macrophage migration inhibitory factor induced-epithelial repair.
Calandra T: Macrophage migration inhibitory factor and host innate immune responses to microbes.
Morand EF, Leech M, Bernhagen J: MIF: a new cytokine link between rheumatoid arthritis and atherosclerosis.
Cvetkovic I, Al-Abed Y, Miljkovic D, Maksimovic-Ivanic D, Roth J, Bacher M, Lan HY, Nicoletti F, Stosic-Grujicic S: Critical role of macrophage migration inhibitory factor activity in experimental autoimmune diabetes.
Stein R, Qu Z, Cardillo TM, Chen S, Rosario A, Horak ID, Hansen HJ, Goldenberg DM: Antiproliferative activity of a humanized anti-CD74 monoclonal antibody, hLL1, on B-cell malignancies.
Kleemann R, Hausser A, Geiger G, Mischke R, Burger-Kentischer A, Flieger O, Johannes FJ, Roger T, Calandra T, Kapurniotu A, et al.: Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1.
Lue H, Kapurniotu A, Fingerle-Rowson G, Roger T, Leng L, Thiele M, Calandra T, Bucala R, Bernhagen J: Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity.


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In Detroit's Black neighborhoods, safety has been sacrificed to austerity.
Real solidarity will help to mobilize forces everywhere that are coming to realize the deepening costs and injustices of accepting capitalism's continuation.
Jason Leopold is the author of the Los Angeles Times bestseller, News Junkie, a memoir. Visit jasonleopold.com for a preview. His most recent investigative report, "From Hopeful Immigrant to FBI Informant: The Inside Story of the Other Abu Zubaidah," is now available as an ebook. Follow Jason on Twitter: @JasonLeopold.

subset: commoncrawl


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5 All-New, All-Different Marvel Titles We're Most Excited to Read
Comic Books
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The Mission
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Spinoff Online
Comics Should be Good
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Robot 6
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CBR TV spoke with Dark Horse Publisher Mike Richardson and the legendary artist/writer Stan Sakai about their new project, a comics adaptation of the true Japanese story of the 47 Ronin. The adaptation has nothing to do with the movie currently in development at Universal, but when he heard it was going to be a movie, Richardson approached Sakai, convincing the artist to put "Usagi Yojimbo" aside so he can work on the historical drama.
Sakai explained that this is a story he grew up with and is one that is truly important to him, marking the first time in his storied career that he's illustrated another artist's work. The art style will be different from what Sakai's fans are used to, depicting humans rather than animals, in an art style inspired by Japanese woodcut. And for traditionalists, Sakai plans to start with a new miniseries when "Usagi" returns from hiatus, before picking up the long-running ongoing title with it's regular numbering.
Awesome video interview!
Great interview! The new book sounds awesome! Stan Sakai is the nicest and most talented guy in comics.
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Listen to free music by Two Pints Of Lager
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Polo Ralph Lauren
Slim-Fit Washed Chambray Shirt
Unfortunately, this item will not be back in stock.
Polo Ralph Lauren offers authentic style, and there are few pieces as classic as the chambray shirt. Cut from pure cotton in a classic blue shade, this button-down collar version is detailed with the label's multi-coloured emblem at the chest for a fresh finish. Wear it with the collar popped over a T-shirt and chinos to give your look contemporary attitude. Shown here with a Sunspel sweatshirt, J.Crew jacket, Polo Ralph Lauren trousers, Tod's bracelet and Quoddy shoes.
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|More News >>
Steve O'dore has been named president and chief executive officer of Indianapolis-based Tangram. He is currently director of behavioral health and life coaching at Tangram and will succeed Connie Dillman, who is retiring in July.
Six Ways To Be 100 Percent... You
I recently spoke at a STEM and women panel at IUPUI for MAEOPP's first ever #GirlsinSTEM Conference... it was a blast, and I met new amazing women! They sent us questions to think about prior to the panel lunch discussion, and I've been noodling on them before, during and after. One was the following: "How do you become or maintain your authentic self?"
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The video and audio excerpts are licensed for private, non-commercial use only. Any other use is strictly prohibited without the expressed, written permission of Grow Indiana Media Ventures, LLC. Duplication in whole or part of the video or audio contained within is strictly prohibited. All other rights reserved.


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Narrow your search
This 23-inch all-in-one is built for entertainment, offering great speakers and a striking look. But the glossy, reflective display throws a wrench in the works.
Whether you're yearning for some '80s arcade action or shopping for a new-school top-down shooter, Galaga Special Edition is a disappointment.
The design could use a few tweaks, but as a waterproof iPhone case, the Tech 21 Submariner will serve you well.
Pricing not available
Dell's Inspiron 17R SE packs reasonably high-end parts into a budget-feeling chassis, saving a few hundred dollars or more over similarly configured laptops.
Pricing not available
HP's new Leap Motion laptop has a built-in motion and fingerprint sensors.
Polarr opted not to build an app specifically for Apple's iOS software, instead going the less-traveled route of using more-universal Web technologies. That decision is now paying off with droves of downloads.
A happy discovery at a certain furniture store gave my workstation a much-needed ergonomic fix.
The 2012 Mazda Miata PRHT Special Edition adds rarity to one of the best drivers' cars of this generation, but the omission of the most basic cabin-tech options severely hurts its score.
Check out the free games of July 2015 available on PlayStation Plus and Xbox Live's Games with Gold services.
The new Xbox One console celebrating Forza Motorsport 6 is extremely blue in color.


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[COLOR=#000000][FONT=Time...sunbuns99 July 3rd, 2015 12:42 AM
[TABLE="width: 450"]
But that's exactly what they did. I should've guessed that Tim, being the stiff...
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[TD="width: 400"]
Speaking of beer, in this same blue tent...
[CENTER][COLOR=#000000][FONT=Times][URL="http://cdn.thedailybeast.com/content/dailybeast/articles/2014/08/14/no-network-for-bisexual-men-and-women/jcr:content/body/inlineimage.img.800.jpg/140800953694...sunbuns99 June 27th, 2015 08:14 PM
Cocky Boys-Seth Santoro Fucks Duncan Blacknataku July 3rd, 2015, 10:33 AM
sgt pepper you are right on point audrey and davone especially DAVONNE is brilliant and she can see people for what they are, but she brings action dramaRRUGER July 3rd, 2015, 10:22 AM
Damn it is it too much for Tomic to stick around the 2nd week! ;((
At least for the pics. his body is perfection. 6'5 is for me...& with an
And, to add to what Sgt. Pepper said above, it'll be another season where the women go after women...that is if either Da or Audrey are their intendedjaysizzles July 3rd, 2015, 10:21 AM
Cormac135 July 3rd, 2015, 10:15 AM
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'The Getaway' Sneak Peek: Chef Jose Andres Golfs With Chi Chi Rodriguez in Puerto Rico (Exclusive Video)
The Esquire Network series follows a revolving cast of famous faces as they explore the world's greatest cities.
Acclaimed chef Jose Andres travels to San Juan, Puerto Rico, for the next episode of Esquire Network's The Getaway.
The series follows a revolving cast of famous faces as they explore the world's greatest cities.
STORY: NBCU Switch-Up: Esquire Network to Take Over Style, Not G4 (Exclusive)
Having recently opened his newest restaurant in Puerto Rico, Andres is ready to experience everything the island has to offer. In this clip, which is exclusive to The Hollywood Reporter, he goes golfing with pro Chi Chi Rodriguez.
He also drinks with the locals and salsa dances in the streets.
The Getaway airs at 9 p.m. Wednesdays.
Never miss a story. Stay up-to-date with THR's Daily Headlines.
Must Reads
Hollywood Insiders Descend on Havana
CBS Entertainment Chief and "Loudmouthed Feminist" Nina Tassler Reveals the Strategy Behind 'Supergirl' Gamble
Paul Rudd and Marvel's Kevin Feige Reveal 'Ant-Man's' Saga, from Director Shuffle to Screenplay Surgery to Studio's "Phase Three" Plans
Comic-Con 2015: THR's Full Guide to Parties in San Diego
HBO's Real-Life Game of Thrones: The Fight to Stay Rich, on Top and Score Bill Simmons
Taraji P. Henson, Viola Davis and Drama Actress A-List Tackle Race, Sexism, Aging in Hollywood
Lena Dunham, Amy Schumer and Comedy Actress A-List in Raunchy, R-Rated Roundtable
Bob Odenkirk, David Oyelowo and Drama Actor A-List on Oscar Snubs, Bad Scripts and Getting Ignored by Robert Redford


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Originally written on Sports Mockery | Last updated 6/9/14
Posted to Chicago BearsChris Conte had a bad season, so much so that Chicago fans sent death threats to other Chris Conte's on Twitter. Including a weather man in Tennessee. Major Wright didn't stand out as much as Conte…but when grading them individually, he was actually worse. Pro Football Focus graded every safety in the NFL for last season, guess where these two finished? Here's a hint: What number was Terrell Owens? That's right… 81. Chris Conte #82 Major Wright #86 According to Pro Football Focus, eighty one other safeties in the NFL played better than the Chicago Bears' starters last season. That includes both starters on every team and roughly their first backup…. yikes. The good news is there's nowhere to go but up. Better news is the defensive line will be much improved next year. Let's hope the Chicago Bears can finish with a more respectable grade at this position next season. 301 total views, 301 views today The post Guess How Many Safeties Played Better Than Conte And Wright Last Season? appeared first on Chicago Fan Board.
PLAYERS: Chris Conte, Terrell Owens
TEAMS: Chicago Bears
TEAMS: Chicago Bears
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ISSUE #4: Spring 2015
"Whenever the worm of panic creeps along your skin, you'll recall the words they taught you in school during National Education sessions: no one owes us a living. At all costs, you must survive."
Paolo Bacigalupi's Yellow Card Man; Kate Osias's Tango; Ng Yi-Sheng's No Other City; Eliza Victoria's Fade; Andrew Cheah's Century of Loneliness; poems by Cyril Wong, Jerrold Yam, Desmond Kon Zhicheng-Mingdé, Michael Gray and Joses Ho.
Past Issues
"All the writing is surprising and fascinating. As a reader, some knowledge of the region helps, but is not necessary. The great thing about LONTAR is the broad range of speculative fiction showcased here with a guaranteed burst of Southeast Asian culture. […] LONTAR has produced quality, intricate and original work that entertains and distills a yearning in the reader for more." —The Review Review
"Learning real things about a region of the world relatively unknown to me is an unexpected bonus of the great stories in LONTAR. With each tale, I was allowed a view through the eyes of someone whose experiences and orientation are so far separated from my own and yet so similar that the journey is wondrous." —Weightless Books


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Surprised by Hope: Rethinking Heaven the Resurrection and the Mission of the Church Pack, Participants & DVD
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Product Information
|Format: Other
Vendor: Zondervan
Publication Date: 2010
Availability: In Stock
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Publisher's Description
Many Christians believe our future in heaven is all that really matters. But that's not what the Bible teaches. Through this small group bible study, Surprised by Hope, premier Bible scholar N. T. Wright brings you inside the Scriptures to grasp the full, breathtaking hope Jesus offers the world, and its implications for your life today.
Gain an exciting new vision for your life on earth in light of your future in heaven. Wonderful as is the promise of heaven, a glorious hereafter is just part of what salvation is about.
What about today? Jesus called his followers the salt of the earth and the light of the world. Your life here and now is of tremendous consequence, and what you believe about the future has a direct impact on how you live in the present.
In six transforming, faith-inspiring DVD sessions, premier biblical scholar N. T. Wright opens your eyes to the amazing full scope of what God's Word has to say about the world to come and the world that is.
This DVD will help you get the most out of each session as you explore such questions as What is heaven really like? Is our main duty as Christians simply to help non-Christians get there? What hope does the gospel hold for this present life? In what ways does God intend for us to experience that hope personally and spread its healing power to the world around us?
This DVD is designed for use with the companion Surprised by Hope Participant Guide (both are included in this pack). Surprised by Hope provides a clearer vision both of the future and of God's kingdom at hand today.
Sessions include:
1. Hope for the World
2. The Hope of the Resurrection
3. The Hope of Heaven
4. The Hope of Jesus Second Coming
5. The Hope of Salvation
6. The Hope of the Church
Author Bio
N. T. Wright is the Chair of New Testament and Early Christianity at the University of St. Andrews in Scotland. He is the award-winning author of many books, including of After You Believe, Surprised by Hope, Simply Christian, The Challenge of Jesus, and The Meaning of Jesus (coauthored with Marcus Borg), as well as the series Christian Origins and the Question of God.
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Healthy Cats
Protect Your Cat and Home from Fleas
In this article
A scratching kitty isn't one you want to pick up -- you don't want her fleas jumping onto you! You can ease your cat's itch and increase your cuddling time with her by knowing how to spot and destroy fleas on your pet and in your house.
Do a Flea Check
Recommended Related to Cats
Your feline will look (and feel!) like the cat's meow after a good grooming session. By nature, cats are extremely fastidious. You've no doubt watched your kitty washing herself several times a day. For the most part she can take care of herself very well, thank you, but sometimes she'll need a little help from you.
Read the Cat Grooming article > >
Before you spend money on flea treatments, find out if these pests have settled on your cat:
- Watch for any signs of movement in your cat's fur. If you spot tiny bugs bouncing off your cat's coat, it's time to fight fleas.
- Comb your cat with a fine-toothed metal flea comb from head to tail several times a day to skim off adult fleas and their eggs. This comb-down will ease kitty's itching, and the pampering will make her purr with pleasure.
- If your cat has fleas, dip the comb in a mixture of water and liquid dish detergent to kill the fleas.
Keep Your Feline Flea-Free
Your cat's warm, furry coat and nourishing blood supply are a flea's dream home. Protect kitty with a flea barrier to prevent these tiny pests from freeloading on your cat.
Products you put on your cat. Spot-on products are safer, more convenient, and more effective than traditional flea collars, dusts, shampoos, and sprays. You can buy them from your vet or on the Internet. Ask your vet where on your cat to put the product, how much of it to use, and how often to use it. If you're not getting the treatment from your vet, read the product label first to make sure it's safe for cats. These are some common active ingredients and brands:
- Fipronil in Frontline Plus
- Imidacloprid in Advantage
- Selamectin in Stronghold/Revolution
Medicines your cat eats. Once a month, you add the flea treatment Program, which contains lufenuron, to your cat's food. It's also available as an injection your cat gets every 6 months. This doesn't kill adult fleas; it keeps them from reproducing.
The pill Capstar, with the active ingredient nitenpyram, kills fleas on your cat within 30 minutes. It does not have any residual effect, though.
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The Griffin Technology PowerBlock Micro is super compact USB AC charger. It allows you to charge your iPhone, iPod touch, nano and classic. It can work for many other USB connectible devices as well. Just plug the PowerBlock Micro into a wall socket and attach your iPod / iPhone via the supplied USB cable or other device by its USB cable. The PowerBlock Micro delivers a quick 5W charge. It is a space-saving, Energy Star qualified unit that is great for quickly charging your iPod / iPhone or other devices that can connect via USB.
- Super-compact 100 - 240V, 5W AC charger for USB devices
- Designed to save energy when not in use, without sacrificing performance
- Includes a detachable USB charge / sync cable for iPod and iPhone


text: A banner is raised over the New Jersey Devils 1995 Stanley Cup winning team as they are honored before an NHL hockey game against the Philadelphia Flyers (AP Photo/Mel Evans) [ Henrique scores 2, Kinkaid makes 25 saves, Devils win By Associated Press March 9, 2015 NEWARK (AP) -- Adam Henrique scored two goals and Keith Kinkaid had 25 saves in his return to the lineup to lead the New Jersey Devils to a 5-2 victory over the Philadelphia Flyers on Sunday night. Read More|Comment Category: Sports Tags: New Jersey Devils, Philadelphia Flyers Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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International Crop Science Conference & Exhibition 2013
January 6-8, 2013(Ended)
Kenyatta International Conference Center (KICC)
Nairobi, Kenya
The International Crop Science Conference & Exhibition 2013 was held in Nairobi, Kenya on January 6-8, 2013.The International Crop Science Conference & Exhibition 2013 is a great opportunity for Agrochemical Manufacturers &...
Harambee Avenue,
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125-250 US Dollar (Early registration date: November 15, 2012)
Abstract submission deadline
January 6, 2013
ImportantPlease, check the conference website for possible changes, before you make any traveling arrangements
* Prices are for evaluation only.
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Physiological response to sun exposure of rams fed sugarcane bagasse silage treated with urea and live yeast culture or pronifer
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Industry conferences and their gains
Serum testosterone, testes size and semen quality of rams fed sugarcane bagasse treated with urea or pronofer


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A daily updated summary of the week in TV.
Series Overview
Executive Producer: Michael Gunton
Genres: Documentary, Nature
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A global view of the specialized strategies and extreme behaviors that living things have developed in order to survive.
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|attached to Users Report Warping of Apple's iPhone 6 Plus
|Re:KIlling off the Microsoft Store Name Too
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|attached to Microsoft Killing Off Windows Phone Brand Name In Favor of Just Windows
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Ring-fencing Italy
Paddy Hirsch
Nov 29, 2011 There's been a lot of talk recently about the need to ring-fence certain economies in Europe. These aren't the economies that have defaulted or been bailed out; these are the economies that are too big to fail. Italy is the most glaring example.
Europe weighs greater fiscal consolidation
Stephen Beard
Dec 2, 2011 Eurozone countries are under pressure to consolidate their fiscal policies as a way to resolve their current debt crisis.
Why the world cares about European bond sales
Stacey Vanek Smith
Dec 2, 2011 The normally prosaic process in which European governments borrow money has become a daily test of their economic health.
Thinking the unthinkable -- a eurozone "divorce"
Sarah Gardner
Dec 2, 2011 If the 17-member eurozone did break up -- either because of bond market pressure or irreconcilable differences -- what would the divorce look like?
Is the euro's death approaching?
Stephen Beard
Nov 25, 2011 In yet another bad development in the eurozone debt crisis, Italy saw its borrowing costs skyrocket. And now a British regulator has called on U.K. banks to prepare for the possible disintegration of the euro.
Financial markets unimpressed by EU treaties plan
Stephen Beard
Nov 24, 2011 Critics say a plan proposed by leaders of Germany, France and Italy to modify European Union treaties won't help with the immediate crisis.
Germany struggles to sell its debt
Steve Chiotakis
Interview by
Nov 23, 2011 The country that's considered the strongest in the eurozone had a hard time convincing investors to lay down cash for its debt today, which means the troubles going on in Europe could be entering a new phase.
Some bright spots in the revised GDP numbers
David Brancaccio
Nov 22, 2011 The revised third quarter GDP figures show 2 percent growth, down from the previously estimated 2.5 percent gain. Few economists were expecting that much of a revision and it sure looks like a disappointment -- until you start picking through the numbers.
PODCAST: What the super committee lost, Egypt's revolutionary flare up
Mary Dooe
Nov 29, 2011 We speak to the President of Panama Ricardo Martinelli about how the debt crises in the U.S. and Europe are affecting his country, and how Panama is continuing to grow. Reporter Mitchell Hartman shows us one place that was shaken to its core by the housing crisis. And what does the OWN network need to do to succeed?
Third quarter GDP revised down
David Gura
Nov 22, 2011 The Commerce Department says the economy grew 2 percent, revised down from 2.5. But economists say chances are after one step back, we'll take two forward.


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The number of grams in 1 cup is determined by the difference between the weight and volume of the substance being measured. For instance, 1 cup of any regular liquid equals approximately 240 grams, while 1 cup of soup containing solid ingredients, such as noodles or vegetables, weighs more.Know More
Ounces, teaspoons, tablespoons and cups are measurements of the volume of substances, while grams measure the weight of a substance. The weight of solids can differ even if substances have the same volume. For instance, 1 cup of flour and 1 cup of white sugar are the same volume, but they are not the same weight. The weight of 1 cup of flour equals 125 grams, while 1 cup of white sugar equals 200 grams.Learn more about Food Measurements
Since a teaspoon is a measurement of volume and a gram is a measurement of mass, it's not possible to convert from one unit to the other without knowing what substance is being used. When comparing teaspoons of different substances, the one that is denser will weigh more.Full Answer >
There are 8 fluid ounces in one cup. One fluid ounce is equivalent to one-eighth of a cup and is a measure of volume as opposed to an ounce, which is a measure of weight.Full Answer >
A cup measures volume and a gram measures weight, therefore the number of grams cannot be given because it is entirely dependent on what is in the cup. A cup of something heavy weighs more in grams than a cup of something light.Full Answer >
A cup of butter is equal to 227 grams, or 8 ounces. In the United States, butter is often measured in sticks, with a cup being equivalent to 2 sticks. A quick way to measure butter is to refer to the printed measurements on the wrapper of an unopened brick.Full Answer >


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Cute alert! Toronto Zoo welcomes five endangered penguin hatchlings
The Toronto Zoo's breeding program pays off as it welcomes its second batch of baby African Penguins. Five hatchlings were born in December.
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The Toronto Zoo announced the arrival of five special, and extremely adorable, new residents on Friday.
Five endangered African Penguin hatchlings join the zoo's rookery of the black and white speckled birds.
The penguin chicks will be on display in their indoor enclosure within the African Savanna display from mid-February after two more eggs in incubation are due to hatch mid-January.
Two of the newborn hatchling's parents, Buddy and Pedro, made international headlines in November 2011 when zookeepers speculated the nuzzling duo were gay. The two were separated and paired with Farai and Thandi respectively and each pair had one hatchling in December.
Their feathered friends DJ and Ziggy had one hatchling on Dec. 10 and Greenbird and Colby produced two on Dec. 20 and 22.
The five December hatchlings are part of the zoo's breeding program that also produced three hatchlings earlier in the spring.
According to the Toronto Zoo website, the African Penguin population is estimated to be less than 10 per cent of what it was in 1900 with about 1.5 million birds on Dassen Island alone. In the early 1990s, an estimated 179,000 adult birds remained and although there was a slight increase in the late 1990s, the annual population declines by an average of 2 per cent each year.
Their largest threats include commercial fisheries and oil pollution.
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text: Willy Wonka And The Chocolate Factory Welcome to the Facotry By WarnerBrosEntertainment On Blu-ray: http://bit.ly/g73vf . A poor little boy wins a ticket to visit the inside... more »On Blu-ray: http://bit.ly/g73vf . A poor little boy wins a ticket to visit the inside of a mysterious and magical chocolate factory. When he experiences the wonders inside the factory, the boy discovers that the entire visit is a test of his character. « less Published October 12, 2009 540 views More Info » Full Credits WarnerBrosEntertainment Uploader Stats & Data 16 Funny Votes 19 Die Votes 538 Views Published October 12, 2009 Categories: Clean Comedy Keywords: Tags Willly Wonka Chocolate Factory Golden Ticket Munchkins classic epic catalog golden film Hollywood legend vintage Up Next: The Vampire Diaries- My Phone AUTOPLAY Up Next In: 10 The Vampire Diaries- My Phone Tom and Jerry: Chuck Jones Collection - Food Moonlight - Good Team Our Gang Collection Follow Facebook Twitter Tumblr Instagram Browse Related Comments 950+ more View All ▶ WarnerBrosEntertainment Vaguely Related Videos Vaguely Related Pics & Words Advertisement Advertisement From Around the Web ZergNet


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Proponents of coconut water claim it can do everything from keep you hydrated to lower your risk of stroke and heart attack. The Nutrition Twins examine the true benefits of coconut water and explain whether or not it might be a good fluid replacement source for people who...
Regardless of what your training goals might be, this functional-training workout will help challenge your body in new ways. These exercises require only your body weight and give you an excuse to enjoy the fresh air as you take your workout outdoors.
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Olivier Schultheis
From Wikipedia, the free encyclopedia
Olivier Schultheis (born April 29, 1965) is a musician, a lyricist, a composer and a conductor. He won the top prize of Conservatoire National de Musique de Paris for his absolute pitch technique.[1]
He became the music conductor in French TV show Nouvelle Star (adaptation of American Idol) and he is now a judge in X Factor on M6 (for the second series in 2011),[2] next to Véronic DiCaire, Henry Padovani and Christophe Willem. His father is Jean Schultheis and his brother is Julien Schultheis.
Albums composer[edit]
- À l'envers, (2004) by Steeve Estatof
- Le Roi Soleil (2005)
- Thierry Amiel (2006) by Thierry Amiel
- Quand l'éternité...(2006) by Hélène Ségara
- Inventaire (2007) by Christophe Willem
- Mon Paradis (2007) by Christophe Maé
- Mozart, l'opéra rock (musical, 2008)
- Best of VO/VS (2010) by Calogéro
- Donne moi le temps by Jennifer
- ^ X Factor page on Olivier Schultheis (in French)
- ^ Olivier Schultheis, le tueur de X Factor? (in French)
External links[edit]
- X Factor website (series 1) (French)
- X Factor website (series 2) (French)
- Olivier Schultheis page at Discogs
|This article about a French musician is a stub. You can help Wikipedia by expanding it.


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2013 Chicago Film Critics Association nominations
I was hit with a pang of sadness this morning when it occurred to me that the Chicago Film Critics Association's list of nominees this year are the organization's first since the passing of legendary Chicago critic Roger Ebert. What would he have thought of "12 Years a Slave," which led the way with 11 nominations? How would he have responded to "Gravity?" What invaluable insight might he have afforded films like "American Hustle," "Her" and "Inside Llewyn Davis," all of which fill out the group's Best Picture slate? We'll never know, and I don't know — it strikes me as particularly unfortunate in a year like this, which has been so, so extraordinary. Rest in peace, Roger. Check out the full list of CFCA nominations below. And remember to keep track of the season via The Circuit.
Best Picture
"American Hustle"
"Inside Llewyn Davis"
"12 Years a Slave"
Best Director
Joel & Ethan Coen, "Inside Llewyn Davis"
Alfonso Cuaron, "Gravity"
Spike Jonze, "Her"
Steve McQueen, "12 Years a Slave"
David O. Russell, "American Hustle"
Best Actor
Bruce Dern, "Nebraska"
Chiwetel Ejiofor, "12 Years a Slave"
Oscar Isaac, "Inside Llewyn Davis"
Matthew McConaughey, "Dallas Buyers Club"
Robert Redford, "All is Lost"
Best Actress
Cate Blanchett, "Blue Jasmine"
Sandra Bullock, "Gravity"
Adèle Exarchopoulos, "Blue is the Warmest Color"
Brie Larson, "Short Term 12"
Meryl Streep, "August: Osage County"
Best Supporting Actor
Barkhad Abdi, "Captain Phillips"
Michael Fassbender, "12 Years a Slave"
James Franco, "Spring Breakers"
James Gandolfini, "Enough Said"
Jared Leto, "Dallas Buyers Club"
Best Supporting Actress
Scarlett Johansson, "Her"
Jennifer Lawrence, "American Hustle"
Lupita Nyong'o, "12 Years a Slave"
Lea Seydoux, "Blue is the Warmest Color"
June Squibb, "Nebraska"
Best Adapted Screenplay
"August: Osage County"
"Before Midnight"
"12 Years a Slave"
"The Wolf of Wall Street"
Best Original Screenplay
"American Hustle"
"Blue Jasmine"
"Inside Llewyn Davis"
Best Foreign Language Film
"The Act of Killing"
"Blue is the Warmest Color"
"The Hunt"
"The Wind Rises"
Best Documentary
"The Act of Killing"
"The Armstrong Lie"
"Stories We Tell"
"20 Feet from Stardom"
Best Animated Feature
"The Croods"
"From Up on Poppy Hill"
"Monsters University"
"The Wind Rises"
Best Cinematography
"Inside Llewyn Davis"
"12 Years a Slave"
Best Original Score
"Spring Breakers"
"12 Years a Slave"
Best Art Direction/Production Design
"The Great Gatsby"
"Inside Llewyn Davis"
"12 Years a Slave"
Best Editing
"American Hustle"
"12 Years a Slave"
"Upstream Color"
"The Wolf of Wall Street"
Most Promising Filmmaker
Lake Bell, "In A World"
Ryan Coogler, "Fruitvale Station"
Destin Cretton, "Short Term 12"
Joseph Gordon-Levitt, "Don Jon"
Joshua Oppenheimer, "The Act of Killing"
Most Promising Performer
Barkhad Abdi, "Captain Phillips"
Chadwick Boseman, "42"
Adele Exarchopoulos, "Blue is the Warmest Color"
Lupita Nyong'o, "12 Years a Slave"
Tye Sheridan, "Mud"
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"We forge the chains we wear in life."
It's a watch for both early adopters and those late to leave the 19th century. Meet the pocketable Apple wearable from designer Tom Ford.
Twins Jacob and Evie Frye lead the Assassin's charge.
The campaign is cooperative and the multiplayer mode has specialists with unique abilities. Read up on the steps Treyarch is taking to keep Call of Duty vital.
This is what Microsoft's suite should cost. Plus: two bonus deals!
The spin-off to hit show "The Walking Dead" goes back to the beginning of the infection that turned everybody into undead cannibals.
Frank and Claire Underwood trust they can rely on your vote on Netflix on 27 February.
A new book about 10 mystery entrepreneurs aims to hook readers with its serial format. Authors Jason Hiner and Lyndsey Gilpin explain why they chose this digital age publishing route.
These gorgeously atmospheric paper dioramas are brought to life with a shadow puppet effect created by a projection technique normally reserved for full scale buildings.
Google is celebrating the 200th birthday of Charles Dickens with a doodle. But the doodle also seems to be trying to get you to use Google Books. Another example of Google's new commercialism?


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|Reviews for My Stolen Child
| Umm chapter 11 . 1/27
It's 2015, are you ever going to continue your story?
| Guest chapter 11 . 6/30/2014
ahh it was so hilarious '' pleasseeeeee continue!
| godess bubbles chapter 11 . 5/1/2014
Well Kaden is Gray so I don't know what house maybe the Snakes, but then again It'll be funny if he's a Lion...Update soon!
| Griffin Raven chapter 10 . 4/20/2014
So if Severus and Lucius were best friends - does that mean he might be the boys godfather or somethng? More important I'd imagine pureblood's having some kind of law against someone stealing/kidnapping a heir or something? Because it would be nice if something very bad or revealing the truth about Dumbledore to wizarding world, inregards to his actions? I'd love to imagine just how Lucius and Cissy would react and feel if they learnt/or were told about how Draco was found by his twin brother Kaden?
| Griffin Raven chapter 7 . 4/20/2014
If Sirius wasn't truly his godfather since Harry wasn't actually truly Harry Potter - then who did Lucius and Narcisssa make Draco and Kaden(?) godfather and godmother if they both have one? Just why would Dumbledore make sure that Harry was blood adopted by the Potters - and how did he do it? Can't truly imagine that Lily would ooperate willingly unless she was either oblivated or memory charmed in someway? But just why would Dumbledore, I mean exactly what would he have to gain by making Harry blood adopted by the Potters and make him the boy-who-lived, fame as his trusted mentor, Potter/Black wealth or something else? As Harry/Kaden isn't a Potter - but what happens to the Potter and Black wealth if he supposedly dies so that Kaden Malfoy can reappear? What about their parents - I mean with them both in prison then who are the boy's guardian?
I love this story and really wish it was either continued or perhaps adopted because I'd love to read about what might happen to all those that had used, manipulated Kaden's life whilst he was "Harry Potter"?
| Griffin Raven chapter 1 . 4/20/2014
Can't help but wonder just how magic would reaact to James's actions inregards to the vaults in Gringotts - I mean would those now that one twin was gone/dead or would it still register him as alive or something? And would the Potter family vaults list this new child as the heir despite him not being born as one or even blood adopted by them? Perhaps Lily should also put a letter in her own vault explaining about the change of apearance of the child known as "Harry Potter" as the child that James Potter kidnapped and that she had no involvement other than perhaps remaining silent (she obviously didn't approve of what he did either!). Would how the Gringotts Goblins, the Ministry would actually treat James Potter legally if they knew/or found out that he kidnapped a baby to replace his son and heir who died?
| Jacquien Esmerelda chapter 11 . 8/29/2013
Love the story! Can't wait for the update
| Lillytha chapter 11 . 8/16/2013
Have you discontinued this story?
| Nilyenea chapter 11 . 7/12/2013
Okay I am so disappoinated that this story has not be updated in forever. Have you just switched over to Sherlock completely? I love where this story was headed and niw just nothing...
| NekoAngel13 chapter 11 . 7/2/2013
Really like the story. In case you didn't know, in canon Lucius is sent to azkaban after the department of mysteries fiasco but Narcissa isn't as she isn't there. Just thought I'd mention it as someone would've had to look after Draco during the summer.
I like the plot and hope you'll come back to it soon :)
| Saturnblue chapter 11 . 4/7/2013
I have to ask if Dumbledore is the one who did the spell on adopted Malfoy heir then did Dumbledore know the current Malfoy heir that shown up is the same one that pretend to die as Harry Potter? Or did he not know that Harry knew about his true inheritance? I wonder what will Mr. and Mrs. Malfoy will said once they got out of the jail and will Voldemort still be insane as ever like in canon or will he be sane and approachable?
It's rather confusing as to whether or not Dumbledore know the current Harry Potter who pretend to die is the same one that is showing up as Kade Malfoy now.
| CEE chapter 10 . 2/28/2013
Please, please PLEASE continue this fic. It is an amazing concept and it would be brilliant to see how it ends.
PS. Slytherin
| sodalover123 chapter 11 . 2/20/2013
Pls update this story! I love this story!
| Lucy-Erza-Hearthfilia chapter 11 . 12/30/2012
ok love the story btw
| Griffin Raven chapter 6 . 10/16/2012
If Lucius & Cissa Malfoy are both currently detained in Azkaban then just who is currently
Draco's guardian or is it his godfather? Also does Harry (oopppss...sorry forgot his true
name?) have the same godfather...and just how will said godfather handle the sudden
appearance of Draco's twin?
More importantly if Harry was stolen from beside his twin brother & clearly isn't related
to the Dursleys at all apart from the illegal blood adoption...then it becomes clear that
that he isn't the "savior" of the wizarding world which would mean that it could only be
Neville Longbottom? Just what will Harry & Draco plan to do about there parents Lucius &
Cissa being in Azkaban...will they consider telling them of the return of the stolen
child they believed died & would this have any affect upon them? Just what is that lemon
sucking meddling old goat Albus Dumbledore up to with this so-called twin disco? Just
how much of the truth about Harry's parentage & the late James Potter's actions does he
know about? At least Harry knows now just why the sorting hat tried to sort him into
Slytherin house?


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Chico Day Care
19 day cares near you in Chico, CA. Join Care.com for free and find the best one for your family.
Chico, CA
La Casita Primera Preschool and Afterschool Program LLC located in Chico, CA is an art and inventiveness-based program with organic food, gardening, play, and cultural, bilingual environment. The scho...
Est. 2011 Established 2011
Chico, CA
Innovative Preschool in Chico, California offers childcare and preschool education. They provide an environment where children's cognitive, social, emotional and physical growth are encouraged with fu...
Est. 1986 Established 1986
Chico, CA
Chico Center For Learning located in Chico, California operates Mondays through Thursdays from 8 AM to 5 PM and offers one-on-one tutoring in Reading and Math for K-8. The center offers an exceptional...
Est. 2008 Established 2008
Chico, CA
Chico Oaks Adventist Preschool in Chico, California is a non-profit organization and dedicated to providing the highest quality services for children and families, and to demonstrating success in clea...
Est. 2010 Established 2010
Chico, CA
Marys Daycare located at 2002 Huntington Drive Apartment 60, Chico, California, offers basic childcare and learning services. It offers learnings for infants, toddlers, preschooler and kindergarten pr...
Est. 2010 Established 2010
Chico, CA
Chico Montessori Children's House located in Chico, CA uses the Montessori method looking unto children's physical, social, emotional, cognitive, and language development. The school opens Mondays thr...
Est. 1973 Established 1973
Chico, CA
Chico Christian Daycare & Pre-School located in Chico, CA offers a carefully supervised and balanced program for children ages two to five. The program includes a wide variety of activities such as mu...
Est. 1971 Established 1971
Chico, CA
Maya Mi Escuelita is a state-licensed child care center that offers early care and learning programs for preschoolers. Located in Chico, California, the company provides its services to children ages ...
Est. 2008 Established 2008
Chico, CA
Parkside Preschool & Day Care is a state-licensed early childhood facility that offers care and learning programs for children ages 2 months old to 9 years old. Located in Chico, California, the compa...
Est. 2004 Established 2004
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Arizona Cardinals
Arizona Cardinals coach Bruce Arians to team: 'We don't need any distractions'
TEMPE, Ariz. -- Often times it seems like the worst place for a football player to be is away from the field.
On the field, the worst thing someone can do is commit a bad penalty. Off it, well, the possibilities are limitless.
So, as the Arizona Cardinals head into their bye week, head coach Bruce Arians had some help in delivering a message to his team.
"We had a great speaker today, a Navy SEAL," he said Wednesday. "Talked about resting or reloading, then the difference between the two.
"Hopefully we reload, come back Monday for a great practice."
Then, Arians added his own message.
"Don't be �the guy,'" he said. "Too many times, when a guy gets a day off he gets tased in Miami, in South Beach or something. We don't want any of that stuff; we don't need any distractions, that was the message."
Throughout the week, Arians talked about how pleased he was with his team's mindset heading into the break. At 4-4 the Cardinals are very much in the thick of the NFC playoff race, and the first-year boss wants to make sure his players come back ready for the second half of the season.
But part of that is avoiding even thinking about the postseason.
"Playoffs, they're months away, and if you start worrying about them you won't be in them," he said. "It's a day-to-day process. We play the Houston Texans next, and the only way to be ready for the Houston Texans is to have a great Monday, then a great Tuesday, then a great Wednesday.
"You control what you do, those 24 hours. Put it to bed and go to the next one, and then you'll just stack them up pretty good on Sunday."
Cornerback Patrick Peterson is taking a similar approach, saying he sometimes peeks at the standings but tries not to focus on them too much.
"Obviously, the last couple years we haven't had an opportunity to do any peeking," he said. "This year, we're at .500 and we want to keep getting some wins and hopefully, later down the road, I can start peeking."
How do they get to that point?
"We've just got to win the games we're supposed to," Peterson added. "We've got a good stretch of winnable games�looking at the schedule, we believe there's no team on our schedule that can beat us if we go out and play Cardinal football."
That means, according to Peterson, playing fast, physical and with relentless effort. They also must be mistake-free.
That includes this weekend, when they will not even be playing a game.
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We were taken there by Japanese friends. It is a older hotel and most of the staff were older as well. There is a huge, fantastic hot spring bath area downstairs with the hot water flowing from pool to pool with various temperatures in each pool. The rough rocky separations can be hard on tender feet. I found several variations... More
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You can now invite anyone to meet at your favorite Starbucks.
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22 year old woman | Elizabeth, CO, USA | Seeking men 18-30 within 50 miles
Lovin' life!
Describe myself? Well I'll start with I'm a burrito of uniqueness. I'm really attracted to someone who can make me laugh and has an easy going personality. Im a movie bug and can talk for quite some time about them, so if you love movies we will definitely hit it off! I'm headed back to school in August for cosmetology, and I am very excited! I work as a dog groomer for now and it brings in the pay checks and I get to be with dogs all day. I'm always up for doing something new, I've had horses for quite some time and I was a competitor that did pretty well! I love four wheeling and camping and I'm not scared to get in the mud or go fish or hunt. I also really love the water, everything about it! I'm a pretty good swimmer and really skilled at the doggie paddle. I don't really have a type, so don't be shy and send me an email! That's why we are all here right? Submitted from mobile
Book club , Camping , Coffee and conversation , Cooking , Dining out , Hobbies and crafts , Movies/Videos , Music and concerts , Exploring new areas , Performing arts , Shopping/Antiques , Video games
Aerobics, Baseball, Bowling, Dancing, Other types of exercise, Running, Swimming, VolleyBall, Walking / Hiking
I exercise 1-2 times per week.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'8" (172cms)
|5'9" (175cms) to 7'11" (241cms)
|Body type:
|No preference
|No preference
|I'll tell you later
|No preference
|No Way
|Social Drinker
|Social Drinker
|Retail / Food services
|No preference
|I'll tell you later
|No preference
|Never Married
|Never Married, Divorced
|Have kids:
|Yes, and they sometimes live at home, No
|Wants kids:
|Not sure
|Someday, Not sure
|White / Caucasian
|No preference
|Christian / Other
|No preference
|No preference
|Some college
|Some college