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Prostate tumor cells, which characteristically metastasize to bone, initiate binding interactions with bone marrow endothelium under blood flow conditions through binding interactions with E-selectin. We hypothesized that E-selectin ligands on prostate tumor cells are directly associated with bone-metastatic potential. In this report, we elucidate the identity of E-selectin ligands on human metastatic prostate tumor cells and examine their association with prostate tumor progression and metastasis in vivo. To our surprise, we found that the E-selectin-binding form of P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on the human bone-metastatic prostate tumor MDA PCa 2b cell line. Interestingly, we also found that human prostate tumor cells derived from bone, lymph node and brain metastases expressed another leukocyte E-selectin ligand, E-selectin ligand-1 (ESL-1). Immunohistochemical analysis of PSGL-1 and ESL-1 in normal prostate tissue and in localized and metastatic prostate tumors revealed that ESL-1 was principally localized to intracellular cell membrane and expressed on all normal and malignant prostate tissue, whereas PSGL-1 was notably detected on the surfaces of bone-metastatic prostate tumor cells. These findings implicate a functional role of PSGL-1 in the bone tropism of prostate tumor cells and establish a new perspective into the molecular mechanism of human prostate tumor metastasis.
The tropism of prostate tumor cells to bone is a common pathologic process (1, 2) and, as evidenced by numerous reports, appears to be regulated by factors inherent to both prostate tumor cells and bone marrow (BM) microenvironment (3-6). The identity of bone-specific factors, which promote bone metastasis of prostate cancer, is currently undefined. It has been suggested that the fate of metastases formation in a particular organ is due to survival factors indigenous to that organ and that the initial seeding of tumor cells occurs by vascular lodgment (7). The presence of circulating prostate tumor cells and the conspicuous avidity of prostate tumor cells to BM endothelial cells (BMEC) suggest, to the contrary, that migration of prostate tumor cells to bone may be mediated through a bone-specific homing mechanism (8-11).
Human hematopoietic progenitor cells (HPC) require the coordinated functional expression ofthe chemokine receptor, CXCR4, and endothelial (E)-selectin ligands for migration to human BM(12-17). While CXCR4 binding to stromal cell-derived factor-1α (SDF-1α) mediates HPC migration across BMEC (13), E-selectin ligands expressed on HPC support rolling adhesive interactions on BMEC that initiate a cascade of molecular events and facilitate HPC entry into bone. HPC E-selectin ligands, PSGL-1 and CD44, are specialized membrane protein scaffolds bearing sialyl Lewis X epitopes (HECA-452 antigen) concomitant with E-selectin-binding activity (14). Recent in vivo homing experiments indicate that PSGL-1 is a major contributor of HPC E-selectin ligand activity (16, 17), highlighting the importance of BMEC E-selectin and HPC E-selectin ligand(s) in bone homing.
In the context of prostate tumor metastasis, SDF-1α mediates trans-BMEC migration of human prostate tumor cells, while CXCR4 expression is elevated on localized and bone-metastatic prostate tumors, implicating this mechanism in the bone tropism of prostate tumor cells (10, 18, 19). Recent data from our laboratory show that BMEC E-selectin helps initiate adhesion of bone-metastatic prostate tumor cells with BMEC and that HECA-452 antigen expression is associated with prostate tumor progression (20). We hypothesize, therefore, that circulating prostate tumor cells utilize a similar bone-homing mechanism as HPC and that acquisition of E-selectin ligand expression may correspond to a bone metastasis phenotype. Though CD44 is a viable E-selectin ligand candidate due to its noted expression on prostate tumor cells (21, 22), other leukocyte E-selectin ligands, such as PSGL-1, L-selectin and ESL-1, represent other potential E-selectin glycoprotein ligands by virtue of their potential expression of HECA-452 antigen (14, 23-25). The identity of E-selectin ligand(s) on human prostate tumor cells are currently unknown and is the focus of our work described herein.
In this study, we investigated the identity of E-selectin glycoprotein ligands on human prostate tumor cells derived from bone, lymph node (LN) or brain metastases. We found that PSGL-1 bearing HECA-452 antigen and E-selectin-binding determinants, otherwise known as cutaneous lymphocyte-associated antigen (CLA), was expressed on human bone-metastatic prostate tumor cells. In addition, we identified the E-selectin ligand, ESL-1, on all metastatic prostate tumor cells. Immunohistochemical analysis of ESL-1 on normal prostatic tissue and on low and high grade prostate tumors revealed that ESL-1 was highly expressed on all prostatic tissue and was principally localized to intracellular membranous structures. PSGL-1 expression, on the other hand, coincided with high E-selectin ligand activity on the bone-metastatic prostate tumor MDA PCa 2b cell line and was directly associated with bone metastases. Immunohistochemical analysis showed that PSGL-1 was conspicuously detected on the surface of prostate tumor cells in bone, whereas low to negligible levels of PSGL-1 were found on normal prostate epithelium, localized prostate cancer and prostate tumor metastases in non-bone tissue. These data are the first described to date and support the notion that PSGL-1/CLA may facilitate the bone tropism of prostate cancer.
Cell Lines. Human HPC KG1a cells and murine monocytic WEHI-3 cells (both from ATCC®, Manassas, VA) were maintained in RPMI-1640 with glutamine/10% FBS/1% penicillin/streptomycin (P/S) (all from Gibco™ Invitrogen Corp.; Grand Island, NY). Human prostate tumor MDA PCa 2b cells derived from bone metastases (26) were propagated in BRFF-HPCI (AthenaES™, Baltimore, MD)/20% FBS/1% P/S. Other human bone-metastatic prostate tumor cell lines, PC-3, PC-3M, PC-3M Pro-4 and PC-3M LN-4 (27,28), were maintained in RPMI-1640 with glutamine/10% FBS/1% P/S, while PC-R1 and PC-E1 cell lines (generously provided by Dr. Klaus Pantel; Hamburg, Germany) (29) were cultured in RPMI-1640 with glutamine/10% FBS, 1% P/S, 10μg/ml transferrin, 5μg/ml insulin, 10ng/ml recombinant human EGF and 10μg/ml recombinant human bFGF. Human LN-metastatic prostate tumor cell lines, LNCaP, LNCaP Pro-5, and LNCaP LN-3 (28), and human brain-metastatic prostate tumor DU-145 cells (ATCC®) were maintained in RPMI-1640 with glutamine/10% FBS/1% P/S. Human BMEC, HBMEC-60 (kindly provided by Dr. C. Ellen van der Schoot; Sanquin Research at CLB; Amsterdam, Netherlands (30)), were cultured in Medium199 with HEPES and glutamine/10% FBS/10% human serum/100μg/ml G418/5U/ml heparin/1ng/ml recombinant human bFGF/1% P/S.
Parallel-Plate Flow Analysis. For cell rolling assessments on E-selectin natively expressed on human BMEC, prostate tumor cells and (+) control KG1a cells were perfused over confluent cultures of HBMEC-60 grown in 35x10mm culture dishes (Corning Inc., Corning, NY) and stimulated for 4 hr with 10ng/ml IL-1β (Sigma Co., St. Louis, MO) prior to assay as previously described (20). To confirm E-selectin expression, cells were harvested with 0.5mM EDTA and stained with anti-human E-selectin moAb 68-5H11 (BD Biosciences, Inc., San Jose, CA) for flow cytometric analysis. IL-1β-stimulated HBMEC-60 cells treated with 10μg/ml neutralizing anti-human E-selectin monoclonal Ab 68-5H11 for 30 min. at RT was performed to confirm E-selectin-mediated adhesion. Prostate tumor cells released with 0.5mM EDTA and washed twice in PBS were suspended at 1x106cells/ml in HBSS/10mM HEPES/2mM CaCl2 assay medium and then infused into the chamber over HBMEC-60 cultures as previously described (20). Cell rolling was assessed at 0.6 dynes/cm2 from the mid-point of the chamber viewing field (4 fields of view, 3 different experiments) at 100X magnification as previously described (31). All experiments were observed in real time and videotaped for offline analysis.
Immunoprecipitations and Western Blot Analysis. Membrane preparations of prostate tumor, WEHI-3 and KG1a cells were prepared, and membrane protein was quantified by Bradford method as previously described (14, 20). E-selectin ligand, PSGL-1 and ESL-1 were immunoprecipitated from membrane protein solubilized in 2% NP-40 and precleared in Protein G-agarose with recombinant murine E-selectin-human Ig chimera (R & D Systems, Inc., Minneapolis, MN), mouse IgG anti-human PSGL-1 moAb KPL-1 (BD Biosciences, Inc., San Jose, CA) and rabbit polyclonal anti-sera against ESL-1, respectively. ESL-1 anti-sera was generously provided by Dr. Bruce Furie (Beth-Israel Deaconess Medical Center, Boston, MA) and was also prepared by immunizing rabbits with KLH-conjugated peptides from amino acids 157-176 of ESL-1 (Invitrogen Corp., Carlsbad, CA). Solubilized membrane protein precleared in protein G-agarose was mixed with antibody/chimera at a mass ratio of 100:4, incubated for 18hr on a rotator at 4°C, mixed with protein-G agarose for 2hr at 4°C, and then analyzed by Western blotting. Immunoprecipitations performed with respective isotype controls were conducted in parallel to control for non-specific protein binding. Where indicated, surfaces of WEHI-3 and prostate tumor cells were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin (Pierce Biotechnology, Inc., Rockford, IL) according to manufacturer's protocol prior to membrane protein preparation. Biotinylated membrane protein was incubated with avidin-agarose (Vector Labs., Burlingame, CA) for 18hr on a rotator at 4°C and analyzed by Western blotting.
For Western blotting, total membrane protein, biotinylated membrane protein or avidin/immunoprecipitates were subjected to reducing SDS-PAGE on 4-20% gradient gels, transferred to Immunoblot™ PVDF membrane (Bio-Rad, Inc., Hercules, CA), and blotted with respective antibody. To confirm that quantified membrane protein was equivalent, identically-loaded SDS-PAGE gels were prepared in parallel and stained with Coomassie blue R-250. Intensity analysis of Coomassie-blue stained protein showed that membrane protein loaded for blotting experiments was identical. Blots were first blocked in FBS and then incubated with E-selectin-Ig, anti-PSGL-1 moAb KPL-1, rat IgM anti-human CLA moAb HECA-452 (BD Biosciences) or ESL-1 anti-sera (all at 1 μg/ml) for 1 hr at RT. Isotype control immunoblots were performed in parallel to evaluate non-specific protein binding. Blots were then incubated with respective alkaline phosphatase (AP)-conjugated secondary Abs (all at 1:1000) (Zymed Labs. Inc., San Francisco, CA) for 1 hr at RT and developed with AP-substrate Western Blue® (Promega; Madison, WI) as previously described (20). These experiments were performed a minimum of 5-times.
Purification and Mass Spectrometry of E-selectin-Ig-reactive 150kDa Membrane Protein. To isolate the 150kDa protein(s), we separated MDA PCa 2b membrane protein on reducing 4-20% SDS-PAGE gradient gels and analyzed the E-selectin-Ig-reactive 150kDa protein as follows. To guide localization, excision and retention of the relevant protein, an E-selectin-Ig immunostained blot was prepared in parallel, and the stained blot was superimposed with the corresponding gel. Excised gel fragments from corresponding gels were loaded into a single well (three gel fragments/well) of a new 4-20% gradient gel and subjected to reducing SDS-PAGE. This semi-purification method was repeated 3-times, and the last excised gel fragment was digested with trypsin and analyzed by MALDI-TOF mass spectrometry (Molecular Biology Core Facility, Dana Farber Cancer Institute), and the NCBInr database was searched for possible peptide matches.
Flow Cytometry. Cells from suspension cultures or from adherent cultures harvested by 5mM EDTA were washed twice with cold PBS/2%FBS and suspended in PBS/1%FBS. MoAb HECA-452, anti-human PSGL-1 moAb PL-2 (BD Biosciences), anti-human CD44 moAb A3D8 (Sigma), ESL-1 rabbit anti-sera, anti-human E-selectin moAb 68-5H11 or appropriate isotype-matched control antibody (2μg/test) was incubated with cells for 30 min. on ice. Following two washes, cells were incubated with fluorochrome-conjugated secondary antibody for 30 min. on ice. After washing twice, flow cytometry was performed using a FACScan apparatus equipped with an argon laser tuned at 488 nm (Becton Dickinson). Cells stained with relevant isotype control antibody were subtracted from cells stained with test antibody to control for non-specific binding.
Immunohistochemical Analysis. For ESL-1 immunohistochemical analysis, we utilized tissue micro-arrays (TMA) containing 4μm sections of 2-mm cores from formalin-fixed, paraffin-embedded normal prostate tissue and prostate adenocarcinoma (Chemicon International, Inc.; Temecula, CA). Prostate tumors with a Gleason score of 2-6 were designated as low grade tumors, and tumors with a Gleason score ranging from 7-10 were designated as high grade tumors. TMA were deparaffinized and rehydrated according to manufacturer's protocol. For antigen retrieval, TMA were incubated in 10mM citrate buffer (pH 6.0) in a steam pressure cooker per manufacturer's instructions (Biocare Medical, Walnut Creek, CA). TMA were blocked in hydrogen peroxide and normal goat serum (1:20), and then incubated with rabbit anti-sera against ESL-1 (1:1000) for 1hr at RT. After washing, TMA were incubated with HRP linked-anti-rabbit IgG (Envision Plus Kit; DakoCytomation, Inc., Carpinteria, CA) for 30 min. at RT. Staining was performed using standardized development times and diaminobenzidine (Dako) as a substrate. All TMA were counterstained in hematoxylin. For semi-quantitative analysis of cell staining, brown-stained tumor cells were enumerated and divided by total tumor cell count per field of view at 200X magnification (0.785mm2) and multiplied by 100 to obtain a percent positive cell staining value. Two 2mm cores (a minimum of 4 fields of view) were examined per prostate tissue specimen. Using isotype control staining as a reference for background levels, cell staining was scored as absent (≤1% positive tumor cell staining), weak to moderate (≤75% positive tumor cell staining) and high (>75% positive tumor cell staining).
For PSGL-1 analysis, TMA were constructed and immunostained as follows:
TMA were generated from 30 rapid autopsies and represent prostate tumor specimens from the prostate gland and from all potential metastatic sites (32). The rapid autopsy program was approved by the Institutional Review Board of The University of Michigan. TMA cores were assembled using the manual tissue arrayer (Beecher Instruments, Silver Spring, MD) as previously described (33). Tissue cores from designated areas were targeted for transfer to array blocks. Tissue cores in triplicate (0.6mm in diameter) were sampled from each representative tissue block and spaced at 0.8mm from core-center to core-center. After construction, 4 μm sections were cut and stained with hematoxylin and eosin to verify histological diagnosis. All data are maintained on a relational database as previously described (34). TMA were deparaffinized and rehydrated and then subjected to antigen retrieval in 10mM citrate buffer (pH 6.0) in a pressure cooker for 2 minutes. For immunostaining, TMA were blocked in hydrogen peroxide and normal goat serum (1:20), and then incubated with mouse anti-human PSGL-1 antibody (clone PL-2) (1:200) for 1hr at RT. After washing, TMA were incubated with HRP-linked anti-murine IgG (Envision Plus Kit; Dako), developed and counterstained as described above.
Semi-Automated Quantitative Image Analysis. A semi-automated quantitative image analysis system, ACIS II (Chromavision, San Juan Capistrano, CA), was used to evaluate stained TMA. For immunohistochemical analysis, proprietary software for the ACIS II device was used to detect and quantify brown staining intensity and then compares this value to blue counterstain representing background. Theoretical intensity levels range from 0-255 chromogen intensity units (CIU). In pilot experiments for this study, reproducibility of the ACIS II system was tested and confirmed by scoring several TMA. The correlation coefficient for these experiments was r2=0.973. Due to tissue heterogeneity and to the potential of false-positive leukocyte staining with anti-PSGL-1, a pathologist electronically circled the areas of interest on each tissue core with the ACIS II software, ensuring that intensity measurements were consistent with selected stained areas. Benign prostate tissue cores were included on each TMA to help normalize each slide.
Statistical Analysis. Intensity values were obtained for each tissue core and then normalized within each TMA. Due to potential variation of immunohistochemical staining and intensity values for each TMA, data were normalized for each TMA and from multiple TMA. Staining intensity values for each tissue core from a given TMA were subtracted by the mean intensity for that same TMA and then divided by the standard deviation:
Where j = 1, …, ni ( ni is the total number of cores on TMAi ) . As a result, each normalized TMA had a mean score of 0 and standard deviation equal to 1. Data were then combined using this normalized scale and analyzed using the SPSS software (SPSS Systems, Chicago, IL).
To analyze candidate E-selectin ligands, we first confirmed the expression of E-selectin ligand activities on several bone-, LN- and brain-metastatic prostate tumor cell lines in the parallel-plate flow chamber (20). We assayed tumor cell rolling on E-selectin natively expressed on BMEC. In an E-selectin-dependent manner, we found that bone-metastatic MDA PCa 2b cells exhibited high rolling activity on IL-1β stimulated HBMEC-60 cultures, while PC-3M LN4 cells possessed low activity and all other cells lines did not support rolling adhesions (Figure 1A). Flow assays performed in the presence of neutralizing anti-human E-selectin Ab completely abrogated rolling activity (data not shown).
We subsequently wanted to determine the repertoire of potential E-selectin glycoprotein ligands on human metastatic prostate tumor cells. Prior studies have indicated that the reactivity of moAb HECA-452 to human HPC membrane proteins on Western blots conferred E-selectin-binding function (14, 23-25). Accordingly, we performed Western blot analysis of HECA-452 antigen on membrane protein prepared from prostate tumor cell lines. As shown in Figure 1B, HECA-452-reactive membrane protein from HPC KG1a cells was represented by CD44 at 100kDa and PSGL-1 at 130 (monomer) and 220kDa (dimer). Interestingly, prominent HECA-452-stained membrane protein from MDA PCa 2b cells was evident from 110-130kDa and at 220kDa. Of note, a single HECA-452-reactive band at 150kDa was detectable on cell lines that did not express E-selectin ligand activity (Figure 1B). To help confirm that HECA-452-reactive proteins corresponded to potential E-selectin glycoprotein ligand(s), we performed Western blotting experiments on membrane protein using E-selectin-Immunoglobulin (Ig) chimera as a probe. Similar to HECA-452 immunostaining analysis, we found that KG1a membrane protein stained at 100, 130 and 220kDa (representing CD44 and PSGL-1) and MDA PCa 2b membrane protein(s) was stained at 110-130kDa with E-selectin-Ig. A distinct protein at 150kDa from MDA PCa 2b cells as well as from E-selectin ligand (−) cell lines PC-R1, PC-E1 and DU-145 was also stained with E-selectin-Ig (Figure 1C).
Since HECA-452-reactive PSGL-1 migrates at 120-130kDa and 220kDa (14), we examined the expression of HECA-452 antigen on anti-human PSGL-1 immunoprecipitate from MDA PCa 2b membrane protein. HECA-452 immunostaining of anti-PSGL-1 immunoprecipitate from KG1a membrane protein clearly demonstrated the expression and gel mobility pattern of PSGL-1 (Figure 2A). To our surprise, we found that anti-human PSGL-1 immunoprecipitate from MDA PCa 2b membrane protein was also reactive to moAb HECA-452 at 130 and 220kDa (Figure 2A). By definition, HECA-452-reactivity of PSGL-1 confers E-selectin-binding activity and designation as CLA (14, 23, 31). CLA is a major E-selectin ligand on human HPC and on human skin-homing T-cells (14, 23, 31). Anti-human PSGL-1 immunoprecipitate from KG1a membrane protein was similarly immunostained with Eselectin-Ig at 130kDa and 220kDa, while anti-PSGL-1 immunoprecipitate from MDA PCa 2b cells was stained with E-selectin-Ig at 120 and 130kDa (Figure 2B). Absence of staining at 220kDa in anti-PSGL-1 immunoprecipitate from MDA PCa 2b cells was also noted, suggesting that E-selectin-binding species were only detectable on Western blots in the monomer form. Staining of isotype control immunoprecipitates at 100kDa and not at 120 and 130kDa with E-selectin-Ig indicated that staining at 100kDa was non-specific and that staining of anti-PSGL-1 immunoprecipitate at 120 and 130kDa was PSGL-1 (Figure 2B). Since all other prostate tumor cell lines did not express CLA, we investigated by FACS analysis whether PSGL-1 polypeptide was inherently expressed on metastatic prostate tumor cells. We found that PSGL-1 polypeptide was variably expressed on all metastatic prostate tumor cell lines (from 5-75% positive) (Table 1). As previously reported, prostate tumor cells express CD44 (21, 22) and thus, we considered the possibility that CD44 could represent HECA-452-reactive membrane protein at 110kDa on MDA PCa 2b cells. FACS analysis of CD44 showed that CD44 expression was high on most metastatic prostate tumor cell lines; however, CD44 was notably absent on MDA PCa 2b cells (Table 1). Collectively, these results showed that CLA expression and not CD44 expression was associated with robust E-selectin ligand activity on prostate tumor cells and that CLA was expressed on human bone-metastatic prostate MDA PCa 2b tumor cells. The fact that CLA expression was noted only on MDA PCa 2b cells among several other bone-metastatic prostate tumor cell lines should not weaken the mechanistic implications of these findings. For example, studies on the identification of E-selectin ligands on hematopoietic stem cells, CD44 (HCELL) and PSGL-1, were performed utilizing a single human HPC line (KG1a), which led to subsequent analyses and confirmation that HCELL and E-selectin-binding PSGL-1 were also expressed on freshly-isolated human HPC (14).
The distinct E-selectin-Ig-reactive 150kDa membrane protein on MDA PCa 2b cells prompted subsequent biochemical analyses to identify this protein. We performed sequential SDS-PAGE purification of the gel fragments corresponding to the E-selectin-Ig-stained 150kDa protein and analyzed resolved protein(s) in the resultant gel fragment by MALDI-TOF mass spectrometry. Estimated molecular weights of trypsin-digested peptides in this gel fragment strongly corresponded to the molecular identity of a sialylated membrane glycoprotein known as E-selectin glycoprotein ligand (ESL-1) (35, 36). ESL-1 is a major E-selectin ligand on murine myelocytes and has high homology to the type 1 sialo-membrane protein of the medial cisternae of the Golgi apparatus, MG-160 (35-37). To confirm the localization of ESL-1 at 150kDa, we immunoblotted MDA PCa 2b membrane protein and membrane protein from ESL-1 positive WEHI-3 cells with rabbit IgG anti-sera raised against ESL-1. We found that ESL-1 expression was evident and co-localized with the E-selectin-Ig-reactive 150kDa protein from MDA PCa 2b cells (Figure 3A). To verify that the 150kDa protein on MDA PCa 2b cells was ESL-1, we blotted E-selectin-Ig-purified protein with ESL-1 anti-sera, and alternatively, blotted anti-ESL-1 immunoprecipitate with E-selectin-Ig. As shown in Figure 3B, ESL-1 was detectable in E-selectin-Ig affinity-purified eluate and not in eluate prepared in the presence of EDTA, which confirmed the requirement of Ca++ for ligand binding activity. Likewise, anti-ESL-1 immunoprecipitate from WEHI-3 and MDA PCa 2b membrane protein was stained with E-selectin-Ig, whereas isotype control immunoprecipitates did not stain with E-selectin-Ig (Figure 3C).
To examine the presence of ESL-1 polypeptide on all metastatic cell lines, we blotted tumor cell membrane protein with ESL-1 anti-sera. We found that ESL-1 polypeptide, representing both functional and non-functional forms, was expressed all cell lines (Figure 4A). However, FACS analysis of ESL-1 was relatively low to absent on most cell lines (0-13% positive), with the exception of WEHI-3 cells (91% positive) and LNCaP LN-3 cells (63% positive) (Table 2). These data suggested that ESL-1 expression as detected by Western blotting experiments may be localized to intracellular membranous structures. To help validate this notion, we performed Western blot analysis of ESL-1 in avidin-agarose purified eluate of surface biotinylated membrane protein. Compared with the presence of ESL-1 in avidin-purified eluate from WEHI-3 cells, ESL-1 was relatively absent in avidin-purified eluate from prostate tumor cell lines (Figure 4B). Results from these experiments indicated that ESL-1 in either E-selectin-binding or non-binding forms was expressed on metastatic prostate tumor cells, and that ESL-1/MG-160 appeared to be an intracellular membrane protein as previously reported (35-37).
To investigate the role of ESL-1 as a marker of prostate tumor progression, we performed immunohistochemical analysis of ESL-1 on TMA of normal prostatic tissue and of low (score of 2-6) and high (score of 7-10) grade prostate tumor tissue. Control staining experiments were performed using paraffin-embedded MDA PCa 2b cell pellets to determine appropriate ESL-1 anti-sera concentrations for antigen-specific detection (data not shown). As summarized in Figure 4C, ESL-1 expression was relatively high on all normal prostate epithelial cells and prostate tumor cells from localized cancer, as 75% of cells stained positive with ESL-1 anti-sera. Furthermore, we observed intense peri-nuclear staining, which was localized to intracellular structures of the Golgi apparatus (Figure 4D). These results in collaboration with Western blot and FACS analysis of ESL-1 on metastatic prostate tumor cell lines suggested that ESL-1 was expressed as an intracellular membrane protein on all normal and malignant prostate tissue and was not associated with prostate tumor progression and metastasis.
Prior immunohistochemical analysis of PSGL-1 has indicated that PSGL-1 antigen is primarily found on hematopoietic cells: PSGL-1 is broadly expressed on immature HPC and on mature leukocytes, including monocytes, granulocytes, dendritic cells and lymphocytes (38). To investigate the expression of PSGL-1 in prostate cancer, we performed immunohistochemical analysis of PSGL-1 on TMA constructed from benign prostate tissue, localized prostate cancer and metastatic deposits from a number of tissues. Preliminary immunohistochemical experiments were first performed on paraffin-embedded MDA PCa 2b and (+) control KG1a cell pellets with anti-human PSGL-1 moAb PL-2 to ascertain relevant dilutions of moAb PL-2 and of secondary Abs and development reagents for PSGL-1-specific reactivity (data not shown). Following immunohistochemistry with moAb PL-2 on TMA, benign prostate epithelium and malignant prostate tumor cells within the cores were identified by histologic examination, and staining intensities pertaining to selected prostatic tissue were normalized and analyzed using ACIS II image analysis. Of note, intravascular granulocytes were routinely detected with moAb PL-2, serving as an internal positive control for moAb PL-2 reactivity. Normalized PL-2 staining intensity data from these experiments showed that PSGL-1 expression was significantly elevated on prostate tumor cells from bone metastases compared with staining levels on benign prostate epithelial cells and on prostate tumor cells from localized cancer (Independent t-test; p<0.009) (Figure 5A). There was also evidence that PSGL-1 was expressed on prostate tumor cells in lung, LN, liver, bladder and soft tissue metastases (Figure 5B). As illustrated in Figure 5C, PSGL-1 expression on prostate tumor cells within bone-metastatic tissue was robust, whereas PSGL-1 levels on benign prostate epithelium and localized prostate cancer were largely negative. Granulocytes in blood vessels were distinctly positive for PSGL-1 (Figure 5C) and PSGL-1 (+) HPC were routinely detected in BM tissue.
We have previously shown that expression of the sialyl Lewis X-like epitope, HECA-452 antigen, is associated with prostate tumor progression (20). HECA-452 antigen expression on prostate tumor cell membrane glycoprotein(s) and not HECA-452 expression itself correlates with the capacity of human bone-metastatic prostate tumor cells to initiate adhesive contact with BMEC through E-selectin-mediated rolling adhesions (20). Since prostate cancer characteristically metastasizes to bone, we have speculated that binding interactions between E-selectin glycoprotein ligand(s) on circulating prostate tumor cells and E-selectin constitutively expressed on BMEC (39) are critical to formation of prostate tumor metastasis. The importance of E-selectin ligand - BMEC E-selectin adhesive interactions in bone tropism is not unfounded, as human HPC homing to bone in part requires the functional expression of E-selectin ligands, namely HCELL and PSGL-1/CLA (14-17). The coordinated expression of E-selectin ligands and chemokine receptor(s), such CCR4 on skin-homing T-cells and CXCR4 on bone-homing HPC, more notably, is critical for tissue-specific migration (40-42). The fact that human prostate tumor cells express functional CXCR4 and CXCR4 expression is related to prostate tumor metastasis to bone strongly suggests that E-selectin ligands may help promote bone tropism of prostate tumor cells (10, 18, 19).
In this report, we investigated the identity of E-selectin glycoprotein ligands on human metastatic prostate tumor cells. Using the parallel-plate flow chamber, we initially ascertained the prevalence of E-selectin ligand activities on prostate tumor cell lines derived from bone, LN or brain metastases. We observed that E-selectin ligand activity was notably robust on the bone-metastatic MDA PCa 2b cell line, whereas all other cell lines exhibited low to nil activity (20). Since we have previously speculated that HECA-452-reactive glycoproteins represent candidate E-selectin ligand(s) (14, 20), we first performed Western blot analysis of HECA-452 antigen on membrane proteins from both E-selectin (+) and (−) cell lines. Western blotting membrane protein with E-selectin-Ig chimera was also conducted to help show whether prospective HECA-452-reactive glycoprotein(s) expressed E-selectin ligand activity. We found that distinct HECA-452- and E-selectin-Ig-reactive membrane proteins from MDA PCa 2b cells were resolved at ~120 and 220kDa as well as at 150kDa protein, which was also detected on cells that did not possess ligand activity. To identify E-selectin glycoprotein ligand(s) at ~120 and 220kDa, we exercised a candidate immunoprobe approach due to the known molecular weights of leukocyte E-selectin ligand PSGL-1 (14). We performed Western blot analysis of HECA-452 antigen and E-selectin ligand expression on anti-PSGL-1 immunoprecipitates from MDA PCa 2b cells and found that the E-selectin-binding glycoform of PSGL-1 was present. Subsequent FACS analysis of PSGL-1 revealed that PSGL-1 polypeptide was variably expressed at low levels on most metastatic prostate tumor cell lines. To identify the E-selectin-Ig-reactive membrane protein at 150kDa, we performed mass spectrometry on a trypsin-digested gel fragment corresponding to E-selectin-Ig-staining region at 150kDa. We found that the molecular weights of digested peptides closely correlated with a known sialo-membrane glycoprotein, ESL-1 (also known as MG-160) (35-37). Western blot analysis of affinity-purified E-selectin ligand(s) with ESL-1 anti-sera and of anti-ESL-1 immunoprecipitates with E-selectin-Ig chimera was subsequently executed to confirm ESL-1 expression and E-selectin-binding activity. Further analysis of ESL-1 expression revealed that all metastatic prostate tumor cells expressed ESL-1 in membrane preparations, whereas ESL-1 expression was negative as determined by flow cytometry. Indeed, ESL-1 immunoblotting experiments using avid-purified membrane protein from biotinylated whole cells strongly suggested that ESL-1 was not be expressed on the cell surface. Collectively, results from these experiments showed that both leukocyte E-selectin ligands, PSGL-1 and ESL-1, were expressed on human metastatic prostate tumor cells. However, only PSGL-1 bearing HECA-452 antigen (known as CLA) (at 120 and 220kDa) expressed at high levels on MDA PCa 2b cells was coincident with robust cellular E-selectin ligand activity. ESL-1, on the other hand, was expressed, either in its E-selectin-binding or non-E-selectin-binding form, on all cells independent of cellular E-selectin ligand activity. It should be noted that the MDA PCa 2b cell line, opposed to all other bone-metastatic prostate tumor cell lines, is unique in its capacity to recapitulate the molecular and growth characteristics of bone metastases from patients with late stage disease (26). Since the synthetic pathway of E-selectin ligands is governed by a coordinated set of glycosyltransferases and a distinct molecular trafficking mechanism that helps target the glycosylation of specific membrane scaffolds, we believe that MDA PCa 2b cells provided a biological tool to help dissect the potential molecular mediators of E-selectin binding to BMEC. The peculiarity of PSGL-1 and ESL-1 expression on cell lines was subsequently analyzed on localized and metastatic prostate tumor cells in vivo.
Immunohistochemical analysis of ESL-1 on normal and malignant prostate tissue showed that ESL-1 was notably expressed at high levels on all prostatic tissue and restricted to intracellular membranous structures. These results paralleled data from other studies showing ESL-1 or MG-160 expression on the Golgi apparatus of human cells (36, 37, 43). MG-160, first identified as a 160kDa sialoglycoprotein of the medial cisternae of the Golgi apparatus in rat brain (44), has been detected as a Golgi membrane protein in several human fetal and adult tissues, including normal and malignant human brain tissue (43, 45). Our prior findings showing that HECA-452-reactive membrane protein contributes 40% of cellular E-selectin activity (20) coupled with evidence of intracellular localization of ESL-1 and of PSGL-1 expression on metastatic prostate tumor cells indicate that ESL-1 may not be a functional E-selectin ligand and that PSGL-1 could be a key contributor to E-selectin ligand activity on human bone-metastatic prostate tumor cells.
To investigate the relationship between PSGL-1 expression and prostate tumor progression and metastasis, we immunostained normal, benign and malignant prostate tissue, as well as a number of metastatic lesions from various tissues. Other groups investigating PSGL-1 expression by immunohistochemical analysis have surveyed both hematopoietic and non-hematopoietic tissues (38) and have shown that PSGL-1 staining is expressed almost exclusively on immature and mature leukocytes from peripheral blood and from within hematopoietic and non-hematopoietic tissues (38). In our analysis, with the exception of granulocytes and lymphocytes in vessels and hematopoietic tissues, we found that normal, benign and malignant prostatic tissues were largely negative for PSGL-1 expression. However, confidence intervals (95%) from normalized PSGL-1 staining intensities of prostate tumor cells in bone metastases compared with staining intensities of epithelia in benign prostate tissue and localized prostate cancer showed that PSGL-1 expression was statistically higher on prostate tumor cells in bone metastases (Independent t-test; p<0.009). In addition, there was PSGL-1 staining on prostate tumor cells from other potential metastatic tissues, which corroborated with detection of PSGL-1 on other non-bone metastatic prostate tumor cell lines. Because metastatic tumor tissues were not traceable to the primary prostate tumors, we could not directly ascertain the potential causal relationship between PSGL-1 expression and tumor metastasis. Whether acquisition of PSGL-1 expression occurs on specialized tumor cells within localized prostate cancer, on circulating prostate tumor cells or on prostate tumor cells within the BM parenchyma is unknown and currently under investigation.
In summary, our studies elucidate potential E-selectin glycoprotein ligands on human metastatic tumor cells and show that PSGL-1/CLA is a major candidate E-selectin glycoprotein ligand on bone-metastatic tumor cells and PSGL-1 expression may be associated with bone metastasis. Coupled with the role of CXCR4 in prostate tumor metastasis, we speculate that bone metastasis of prostate cancer is a patho-biological outcome, which mirrors the process of HPC homing to bone. Interestingly, we found that E-selectin-binding form of CD44 (HCELL) was not expressed on metastatic prostate tumor cells as previously described on HPC (14). CD44, in the context of prostate tumor metastasis, most likely functions as a hyaluronic acid receptor mediating firm adhesion with BMEC (21, 22). The hyaluronan-binding feature of CD44 has previously been found to play a role in the trafficking of human CD34+ stem cells to BM (46), which further argues the mechanistic similarities between bone metastasis of prostate cancer and HPC homing to bone. Other adhesion molecules mediating prostate tumor cell binding interactions with BMEC and with BM hematopoietic, osteogenic and stromal elements are undoubtedly also required for seeding, retention and growth of prostate tumor cells in bone (22, 47-50). We believe that broadening our understanding of the "HPC mimicry" or bone-homing behavior of prostate tumor cells will help expand our current view of the pathogenesis of bone metastasis. In vivo mouse experiments tailored to examine the homing efficiency of E-selectin ligand (+) or (−) human metastatic prostate tumor cell into human bone will need to be performed to fully appreciate the functional role of E-selectin ligand(s). Information from these analyses will instigate the development of new therapeutic strategies for inhibiting the bone tropism of prostate tumor cells.
We would like extend to special thanks to Dr. Glenn Bubley (Department of Medicine, Beth-Israel Deaconess Medical Center, Boston, MA) for his helpful criticism of pilot experiments, and Dr. Mirna Lechpammer (Children's Hospital, Boston, MA) for her technical assistance in preliminary studies. This work was supported by NIH grants, NCI 1R21 CA102913-02 (C.J. Dimitroff), NCI 1R21 CA104828-01 (C.J. Dimitroff), NIAMS Harvard Skin Disease Research Center Core Grant 5P30 AR042689 and the Prostate Pathology Core of the Dana Farber/Harvard Cancer Center Grant (CA06516-37). The rapid autopsy program was supported by Specialized Program of Research Excellence in Prostate Cancer (NCI Grant CA69568) and The Prostate Cancer Foundation.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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April 24-30 is Medical Fitness Week. During this week, the William G. Rohrer Center for HealthFitness will host the MOVE150 program, which focuses on encouraging our members to be active regardless of your current fitness level. Pick up a tracker sheet at the Member Services Desk, and complete the activities listed until you reach 150 […]
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Rejuvenation facials
Everybody loves the sunshine! Limited exposure is good for the soul and the immediate effects it has on the skin such as a tan and healthy glow are much desired. However, in the long term, sun exposure decreases the production of collagen, which in turn causes damage to the skin making it feel wrinkled and coarse and looking red and tired. Skin rejuvenation treatments will improve skin tone, texture, refine and refresh the skin by increasing the production of collagen in the skin.
1 session full face – $250
1 session full face & neck – $350
Purchase 3 and receive the 4th free.


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- You are here:
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What's it good for? Bloating
Why? Asparagus helps to reduce water retention and bloating by making you wee more.
What else helps? Parsley, celery, tea and coffee all have the same effect.
More ways to beat bloating
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- Breads: Best and worst loaves revealed
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2" Silicon wafers
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Dear Famularo & Associates:
I am a mother of two children whose divorce was final last year. Their father has no visitation with the kids, because he is too busy with his new life. I have a court order that he is supposed to pay me child support, but he either pays me late, or not at all. Is there any way that I can force him to pay his child support automatically without having to beg him for the money every month? My divorce was in San Diego county, but I now live in Riverside County? Does this make any difference? Do I have to take him back to court to get my money?
Dear A.R.:
If you already have an order for child support, you do not need to go back to courts to get his to pay you. What you need to do is collect the child support you are already owed- both the past due support and future child support. To collect future support, all you need to do is file a wage assignment with the court (it does not require a court appearance, it only requires you to prepare and submit a form to the court for its signature), and then serve it on your ex-husband's employer. The employer must then pay you your child support before your ex-husband gets paid. Collecting the past due child support is a little more complicated and requires the help of a family law lawyer. By the way, because your case was originally filed in San Diego county, you will always return to the same courthouse in that county should you ever need to return to court.
-Famularo & Associates
Tuesday, April 8, 2008
How do I enforce my child support order?
Posted by Temecula Family Law Attorney at 10:42 PM
Labels: child support, collecting, family law, lawyer, past due, support
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I feel like the week is off to a great start with an early rain nourishing the plants and feeding our souls.
27 July 2009
4 comments:
mmmmmmmmmmm rain.........ReplyDelete
mmmmmmmmmmmmm you're right as rain =DReplyDelete
I agree with Jennifer, mmmmmmmmm rain. It was 101 and very dry here today, more of the same tomorrow. Mmmmmmmm rain....ReplyDelete
Oh, SQ -ReplyDelete
They're saying that it's "El Nino" again. That means we're getting your rain. It'll change...suddenly, sometimes. Do a rain dance. Plan a water-catching basin that'll be ready when the inevitable storms come. Have fun! Chat soon.
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Gently sloping sides safely protect child during changes. Caregiver faces baby and does not have to change from the side. Portable and can be used on any surface. Size: 6"h.
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Interested in having us manage your event? Have questions about attending or sponsoring an event? Want to give some feedback or offer a suggestion for future events? We'd love to hear from you!
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text: Niece of Baron de Calvadorr and protagonist of Dark Stars & Steam. Retrieved from "http://kinginyellow.wikia.com/wiki/Lady_Constance_de_Calvadorr?oldid=8119" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Characters Add category Cancel Save


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This is a new fighter I have finished recently. It has a 5 1/2″ long 5160 steel blade with phosphate finish and a Japanese inspired cord wrapped, resin soaked handle with a damascus steel menuki.
This knife is availabile for immediate delivery for US$ 175.00, plus US$ 20.00 for worldwide shipping. A kydex sheath is included.
My computer is still down so it is easier to reach me by Facebook Messenger or mobile whatsapp (00-55-15-997120993). Or you can write to firstname.lastname@example.com but it may take me a day or two to get back to you.


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Arthur Hills AS Media Portfolio
Tuesday, 5 January 2016
Home By 7 (Final Cut Continuity Task)
Here is my Final edit of our groups continuity task. I made a couple of improvements from the first draft such as editing the volume of music and editing the contrast and brightness. In my next blog post I will go through how I made these changes. I will also follow this post up with a detailed evaluation.
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Blog Post
Understanding the one-stop-shop principle
This blog analyses the recent guidance on the one-stop-shop issued by Article 29 Working Party and its practical implications.
Blog Post
New Spanish data protection law in 2017?
The Head of the Spanish Data Protection Authority (AEPD) has announced that a draft bill to reform the current Spanish Data Protection Act (LOPD) is in the works for early 2017. With GDPR coming into force across the EU in May 2018, why is Spain doing this? What does this mean for controllers and processors operating in Spain? and what can be expected from this reform?
Blog Post
Getting to know the GDPR, Part 10 – Enforcement under the GDPR – what happens if you get it wrong?
The General Data Protection Regulation ("GDPR") introduces many new obligations for companies (be they controllers or processors) and the question on the minds of most companies is "…and what happens if we don't comply or get it wrong? What is the risk?"
Blog Post
WP29 Guidance on the right to be forgotten
On 26 November the Article 29 Working Party ("WP29") issued WP225 (the "Opinion"). Part I of the Opinion provides guidance on the interpretation of the Court of Justice of the European Union ruling on
Blog Post
Beware: Europe's take on the notification of personal data breaches to individuals
Article 29 Working Party ("WP 29") has recently issued an Opinion on Personal Data Breach Notification (the "Opinion"). The Opinion focuses on the interpretation of the criteria under which
Blog Post
History in the making: the first 'cookie rule' fines in Europe
On 14 January, the Spanish Data Protection Regulator (the "Spanish DPA") issued its first fines for infringement of Spain's implementation of the EU's "cookie consent" requirement. The decision (in
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Position of Spain on the General Data Protection Regulation: flexibility, common sense and self-regulation
As expectation and concerns rise whilst we wait for the final position of the LIBE committee and the European Parliament on the General Data Protection Regulation (the "Regulation"), the report issued
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Cookie consent – developments in Spain
Last month, Spain became the most recent territory to implement Europe's 'cookie consent' requirement through amendments to its law on the Information Society and Electronic Commerce (34/2002).The
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New model clauses for data processors on their way?
We all know that the times when data processors could try to shield themselves from data protection compliance by arguing that the law did not apply to them are long gone.In recent years the role of


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Wood Fireplace Blower Kit
Wood Fireplace Blower Kit
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Wood Fireplace Blower Kit - is a free Complete Home Decoration Ideas Gallery posted at . This Wood Fireplace Blower Kit was posted in hope that we can give you an inspiration to Remodel your Home. This article can be your reference when you are confused to choose the right decoration for your home. This Wood Fireplace Blower Kit This maybe your best option to decor , because having a home with our own design is everyone's dream.
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This is the January issue of Let's Find Out about hibernation. Snow is the best compositional aspect when designing these see-through issues.
- Home
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- Sketchbook
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- Portfolio: The Wild Life of Elk
- Portfolio: Henry the Impatient Heron
- Portfolio: Annie Jump Cannon, Astronomer
- Portfolio: Black Beauty
- Portfolio: Big Cats
- Portfolio: Animal Atlas
- Portfolio: Animals Other Styles
- Portfolio: Animals
- Portfolio: Macarooned on a Dessert Island
- Portfolio: San Diego Zoo Centennial
Thursday, February 4, 2010
Let's Find Out-January
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Every piece of Dune Jewelry is handmade with love and sandy hands here in our Massachusetts studio. We'd like to invite you into the studio to take a closer look at how your memories are captured in our unique designs. This week we feature our Petite Sandbar Earrings.
The pieces are filled so we are able to sand them down to our signature Matte Finish, exposing a mosaic of sand.
Once the pieces have been filled, sanded & polished it's time to package them up. Every piece gets it's own label and care instructions to make sure you keep your piece looking it's best. Then it's sent off to it's final destination, these are going to a lucky customer all the way in California.


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WeeklyBeats.com / Music / r2me2's music / Deboned
Busy week. Needed to help family during most days, so there was less time to write. I improvised with a few ideas I've been thinking about for this track.
This submission is licensed by author under CC Attribution Noncommercial No Derivative Works (BY-NC-ND)
This works. I'm especially liking the left-hand piano chord progression.
Awesome, interesting work


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Hoop Dance is today's modern version of our favorite childhood activity hula hooping. While hula hooping consists of circling the hoop around one's waist in the same direction, Hoop Dance is a full body workout where the body moves in all planes of motion. Many people who have claimed "I can't hula hoop" find that after learning basic Hoop Dance technique that they can indeed "hula hoop" and so much more!
Come get in the Hoop!
Hoop Dance has numerous benefits for the body, mind, and spirit:
- burns calories while providing a great cardio-respiratory workout
- builds muscular endurance and tones entire body
- builds core strength
- improves balance
- improves overall coordination, particularly hand-eye coordination
- improves flexibility
- improves joint mobility and articulation
- can help decrease back pain
- improves dexterity
- helps mobilize the pelvis and sacrum
- improves motor skills
Mind & Spirit
- develops a sense of rhythm
- induces smiling and laughing
- releases stress
- increases ability to focus and to be present
- helps establish a mental and emotional connection
to the body
- improves self-esteem
- promotes a sense of freedom and creativity
- inspires a sense of synergy
- pushes mental boundaries in a positive way
- helps to embrace one's self and others
Hoop Dance Demonstrations
Susan is available for hoop dance demonstrations and lessons at your next party! Contact for pricing and details.


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Vakku Pookkum Kalam | വാക്കു പൂക്കും കാലം | Episode VIII - March 9, 2017
Meet the Diplomats: B&R Opens Another Dimension for South-South Co-ops - March 9, 2017
Li's Hong Kong remarks send strong signals for region's next chief, say analysts - March 9, 2017
Bold 'armless' women provides for her family - March 8, 2017
Israel uncovers online weapons smuggling cell in West Bank - March 8, 2017
Muhyiddin: We'll wait for the final version of Hadi's Bill - March 8, 2017
Sthree Sakthi Award 2017 Award ceremony Part I - March 8, 2017
Sthree Sakthi Award 2017 Award ceremony Part II - March 8, 2017
C D Sarawathi won Sthree Sakthi Award 2017 - March 8, 2017
Muhyiddin: Don't exploit diplomatic row for political mileage - March 8, 2017
South Korean President visits military command post
South Korean President Park Geun-hye on Friday visited the military command post in Yongin. Park was briefed on the situation of military readiness. Park also presided over a National Security Council meeting on Thursday after the DPRK fired a projectile towards South Korea. The president has ordered the military to retaliate following any additional provocations.
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Van Halen Photography
With their 1978 self-titled debut, Van Halen redefined hard rock while Eddie Van Halen's revolutionary techniques forever changed the sound and significance of the electric guitar. The original lineup—frontman David Lee Roth, guitarist Eddie Van Halen, drummer Alex Van Halen and bassist Michael Anthony—dominated the late 1970s and early '80s with hit singles like "Runnin' with the Devil," "Jump" and "Panama."
When faced with the prospect of Roth going solo, they regrouped with former Montrose singer and solo star Sammy Hagar, who helped them evolve in the more melodic rock direction of the time. In fact, the new lineup soared just as high thanks to the hits "Why Can't This Be Love," "When It's Love" and "Right Now" well into the '90s.
In the late '90s, Extreme's Gary Cherone took over vocalist duties—with lukewarm reactions from both critics and loyal fans. But that didn't stop Van Halen from being inducted into the Rock and Roll Hall of Fame in 2007. Shortly thereafter, Roth returned—alongside Eddie's son Wolfgang on bass—and the band resumed selling out arenas all across the globe.
Van Halen
by Andy FreebergStarting at $1,200
Van Halen
by Al RendonStarting at $300


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17 September, 2012
GOSH Cosmetics Long Lasting Lip Marker.
I've really been loving their new BB Cream (Review soon) and the improved eyeshadow trios too, the pigmentation compared to before is great. I have also really been enjoying these two little gems, the GOSH Long Lasting Lip Marker*. These, as you can see, are like marker pens. Apply them to dry lips, let the colour dry and it stays on for about 6 hours. These do work, but you do have to make sure that you apply them to your dry lips, atleast I do.. On wet lips the colour doesn't really transfer or show.
I have the colours Soft Rose and Natural Brown - I wasn't so sure the brown would work, but as the name suggests it does look really natural on. These have a sweet taste when you initally apply them, but once they settle it doesn't feel like you are wearing anything atall.. They wear off well, without looking all gloppy or gathered at the corners of the mouth, like some stains can.
These are just £6.99 each and available from Superdrug stores and online.
. . . I'm Sophie. I've been writing my Beauty Blog and making You Tube videos since 2008. I love all things beauty related and I just love to write about products, show my makeup looks and post reviews. Enjoy!
5 comments:
I've never really got on wit felt tip style lip stains but the pink colour is gorgeous. xoReplyDeleteReplies
They're not the best,smoothest application, but I do like the light stain they give. :)Delete
You should review LipSense! I was on a mission to find the best long-lasting lip colour and this was by far the best one I tried. It's a liquid lip colour that you apply gloss over, I don't know for some reason I really don't like the marker concept (but the pink colour is lovely). LipSense comes in like a million shades, so I love the variety and that it actually works (stays on me for 8 hours!)Just a little tip from one makeup lover to another :)ReplyDeleteReplies
Thanks Chloe, That sounds pretty cool. I will look into it!Delete
- Anonymous25 January 2013 at 05:47
thanks for share.ReplyDelete
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Starring Robert Newton, Alec Guinness, John Howard Davies. (U)
There are numerous versions of Dickens's classic tale – including an animated one from 1974 with the Monkees' Davy Jones doing the voice of the Artful Dodger – but nothing beats director David Lean's (Lawrence of Arabia) black and white adventure from 1948. Oliver (Davies) is the orphaned boy in Victorian England who runs away from a workhouse and falls in with a gang of London pickpockets, including the Artful Dodger (Anthony Newley), who are under the supervision of nasty Fagin (Guinness).
On its original release, the film was accused of anti-Semitism owing to Guinness's harsh portrayal of Fagin (and, if nothing else, it will give younger viewers nightmares) and briefly banned in the US, but the actor gives a mesmerising performance that is well worth catching. It's a dark film, though, and adults should note that some of the themes (there's a murder, and the film opens with Oliver's mother's death) aren't suitable for younger eyes. In 2005, Roman Polanski directed an adaptation of the Dickens story, with Ben Kingsley as Fagin, Jamie Foreman as Bill Sikes and Barney Clark as Oliver, which is enjoyable but not particularly memorable.
As mentioned in the review, the murder of a main character, Oliver's mother's death and the characters of Fagin and Sykes are all disturbing. This version of the Dickens' tale isn't suitable for under-8s despite its 'U' certificate.


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6000 Baydy Peak Road
Osage Beach, Missouri
Contact Us for more information
or a personal tour of property
McNally Properties
4821 Osage Beach Parkway
Suite 204
Osage Beach, MO 65065
Phone: (573) 348-2121
Real Estate Listing Firm:
Over six acres of commercial lake front property with approximately 440 +/- lake frontage, located in Camden County just outside of Osage Beach at the 21 mile marker. Currently, the property is being used as a dock manufacturing company, but can serve multiple uses. Price is for the real estate and the business. Buyer will get all trade names, fixtures, inventory & equipment/trade fixtures, assets and current contracts as well as past clients list. Multiple buildings on site used for different aspects of the business such as welding, mixing concrete, manufacturing and so forth. Fencing around property for security. Owners have decided to retire and are leaving the area.
|Property Features
Date Photo: 2017-05-12 21:24:40
Date Recap: 2017-05-12 21:24:40
Listing Agent ID: 50758301451
Listing Board: 5075
Mile Marker: 21
|Parcel 1: 08-3.0-05.3-000.0-001-011.000
Photo Count: 1
Road Frontage: 722
Spec. Assmt. Year: 2017
Tax Real Estate: 1840
|Tax Year: 2016
Water Frontage: 440
Year Built: 1996
Return to Previous Page www.RealEstateOzarks.com Detailed Property Search


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The Peppercorn, a speciality restaurant, pays homage to the humble Kebabs and Grills, and serves up unlimited...Know More
Orange County Resorts & Hotels Ltd.
The 'restaurant for two' offers you a completely private and elevated dining experience that towers above all else! The Machan is your chance to commune with nature, even as you discover each other in the gentle intimacy of a treetop gourmet meal.
The Peppercorn, a speciality restaurant, pays homage to the humble Kebabs and Grills, and serves up unlimited...Know More
Fine dining for vegetarians comes of age in the Plantain Leaf, which is set amidst verdant coffee and spice...Know More
For starters, we discover the way to a man's heart is through the portals of the generously stocked Granary, a...Know More
The peppercorn deck built on the lake at the resort, is a very scenic and romantic venue where you can dine ...Know More
A dinner laid out exclusively for the two of you at the Infinity Pool deck by the lake at the resort. ...Know More
An exclusive dining experience by the poolside in your own Kodava style Private Pool Villa. Sit back and...Know More
While we have evolved over the years, our spirit has remained the same. Today, as we prepare to expand the locational footprint of our resorts further into India, Africa, and Asia, we have found the need to rename ourselves to reflect our spirit.
We hereby reaffirm that Orange County Resorts & Hotels Ltd, the parent company of the 'Evolve Back' brand, continues to be owned by the House of Ramapuram. The management and operations of the resorts remain the same. Our people and values are the same. Our resorts and holiday experiences at Coorg, Kabini, and Hampi remain the same. We remain rooted to our philosophy of 'Spirit of the land'.
To read a personal letter from our Chairman on the name change.


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Khloé Kardashian's Good American Announcement
Creator of the brand Good American, Khloé Kardashian announced on Instagram that the brand is releasing season two of the collection. She captioned this photo, "We are releasing another drop from Good American season 2 TOMORROW at 9am PT on goodamerican.com. Can't wait for you ladies to rock The Pencil skirt—sexy, comfortable and makes your curves look amazing!! #GoodSquad #GoodAmerican". It will be available online Thursday the the 16th, be sure to check it out!
Photo/Instagram: Khloé Kardashian @khloekardashian
Twitter: Khloé Kardashian @khloekardashian
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Singapore :
Bukit Brown Cemetery
Bukit Brown Cemetery, also known to the local community as Kopi Sua or Coffee Hill, was a public Chinese cemetery that had been established in the early 20th Century. It was the biggest Chinese graveyard outside China.
hotels nearby
map and directions
food and drinks
more information


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Whenever I read those tutorials about reviewing products the all have this basic format.
- Introduce the product by discussing a horrible situation where this product could be helpful. The typical one in online marketing goes along the lines of: "Another early morning and you head to your PayPay to check for any commissions. Your hand excitedly hovers over the login button. You close your eyes as the page loads. The page pops in and… nothing again."
- Talk about how you know how they feel. "I was like you once."


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(This observe was in black and white for USENET's chitchat.religion.misc in breakneck December 1986, in answer to a sue for for information on paganism.
It fit my definite orthodox of tone - that is, a hefty fit into of praise, even from population who when all's said and done take on I'm an asshole - so I supposition some population indoors might employ reading it.)
Paganism is a negligent word for the great people of polytheistic, shamanistic, and mystical non-monotheistic religions. Paganism exists in all cultures, from paleolithic to nominal, but has historically waxed and waned. The ancient Egyptians are an extraction of a rise pagan society; so are the ancient Romans; and all paleolithic cultures from the Old Brickwork Age to the facet control strong pagan elements. An extraction of a less pagan culture would be the West for the stickup thousand years or so, for example the centuries subsequently the Fall of Rome. The control of the Median East by Christians and Moslems has in the same way nearly all familiar out paganism.
Rep of paganism is a accord for other pagnistic principles, even individuals that equitably fight one's own. Such persecutions as control been directed in opposition to paganistic religions by each other are by-products of sponsor struggles and import community exercises impartially than ideologically stimulated. The extraordinarily is to some stretch true of breakneck Judaism, which was the check beneficiary to the traditions of a overpoweringly pagan progress. A slave bank allegedly led to a few hundred thousand slaves with no place to live; to get them, they butchered the associates of pagan cities and took up private residence in the cities themselves. They invoked their war god to let off this action.
Just as, because the early years of the modern Greek mythology were laid down, it was as a after effects of invading Northern barbarians supplanting the older (and to a certain extent gynocentric) Titan mythology with their imported religion, which grew in addition well-bred and less confrontational when on, as happened with Judaism.
Via it came under the thumb of monotheism, the West was conquered by the rise passable Roman culture. The Roman Republic and Rule were characterized by an inexplicably great fit into of religions together in a personal unreserved whole, unremittingly sharing out the extraordinarily geography and even the extraordinarily temples. This vividly eclectic (or "syncretistic",
as it is in addition when all's said and done frequent in studies of the Romans) synthesis is in addition compatible to modern neo-paganism than any other form of forgotten paganism I know of. Static, it finished after the Christian emperors took ended and Rome knock over.
The post-pagan West veteran loyal resurgences of paganism in poles apart forms. If we go out with this at 1000 CE for friendship, we see chief the Inquisitorial epoch, anywhere paganism was punished with death and agony. Then here comes the Rebirth, in which pagan symbolism and principles in art and philosophy were to a certain extent in addition everyday than vividly Christian ones. The Rebirth lasted until the 16th century. Application that the Inquisitions lasted precisely until the Enlightenment epoch, and were bad trendy the Rebirth, but ceased to be totally ideologically stimulated after the chief three centuries.
The Inquest had become a sponsor arm of the Vatican, a stab well-behaved in oodles ways other than suppressing heresy. It all gone much of its time accomplishing sponsor, antifeminist, and undercover goals of the Church. We see in the trial of the Templars in the fourteenth century that uncommonly resolute population were caught in a activist sponsor hostility between the Sovereign of France and the Pope. They were as usual painful, the typical encouraged confessions were perfect, and they were executed, for reasons having zero to do with view or heresy on the contrary as excuses.
It is in the same way trendy the Rebirth that we begin to control register of what we may assume vividly devoted paganism once more. Highest of the grimoires we control go out with from this era; alchemists, repeatedly freely Christian but employing pagan symbolism and texts, were top figure everyday trendy the Renaissance; the Kabbalah and Tarot mutiny in the Rebirth, forming the resolve of modern pagan symbolism. The Rebirth in the same way saw the stub out birth of a revitalization, in excellent form, of Greek humanism, lawfully pagan what of its disguise by Christian Rome and its use of theistic symbols.
The Reorganization was once more a less pagan period; Protestant rulers elation Elizabeth and James carried out their own anti-heresy pogroms, annihilating top figure register of witchcraft. Of well thought-out attract in the Reorganization is Scot's "The Discoverie of Witchcraft", which presents the humanist and rationalist viewpoint on witches which has by and large triumphed today: that witch accusations were in addition repeatedly encouraged by factors such as repulsiveness, particularized bitterness, sternness, and so forth than on ideological aim, and that in fact here were no witches. This is perhaps true very soon of the when Inquisitorial epoch. Prior to on, the Inquest entirely did help in the temporary stamping out of paganism; so if pagans are witches, here were witches.
We require not tizzy much with Murray's allegedly anthropological study of English witchcraft in the Inquisitorial epoch, on the contrary to note that it has been devoutly accepted by oodles modern pagans, and to upfront out some of its flaws. Based on overdue Inquisitorial register and the evenness of the confessions obtained by the Inquistors, and tossing in some disjointed scraps of English folk history and report, Murray asks us to esteem that a paleolithic subculture lasted in England, living semi-naked in the polish, until a number of the beginning of the Reorganization at lowest amount, and possibly until the topical day. Of course overdue Inquistorial confessions were consistent; they were believably dictated to the agony point. A much advanced piece of the design of paganism to Christianity in the past and trendy England's post-pagan epoch is Jessi Weston's classic "From Check to Romance".
Its conclusions were lesser from decades of clear study of the Grail mythology and its anthropological, mythological, and unreserved context.
As a send-off note on the Reorganization, we may note the confess event of patio astrologers and alchemists and their ilk, the top figure eminent examples anyone the sorcerer John Dee and the seer Edward Kelley under Elizabeth. These were the inheritors of Paracelsus and the other alchemists and Christian alleviate doctors, using pagan symbols and methods with a cloak of Christian symbolism. Kelley bunged the work of Dee and Kelley under incomprehensible circumstances; he is understood to control been told by the angels to form a group sex put with Dee and his wife, which they allegedly did for a while; in out of the ordinary disc, Kelley was encouraged from the work by a idea of a new age daylight, which was heresy.
So, on to the Enlightenment of the seventeenth century. This was in addition humanistic than devoted, conversely humanism is a religion on substitute Tuesdays; it all depends which of the oodles polite definitions you use. In any exterior, the seventeenth centuries saw the chief applications of the reformed Greek humanism that originated in the Rebirth. The counter-Christian topical was giving out stronger; in addition and in addition, population were beginning to bill tone with treatment for all, and independent lifestyle from the get hard area of supposition and participation imposed on them by governments and churches the same. This humanism has painted top figure "ill will" devoted exercises in America for example this time, much for the advanced in my sympathy. This is what training of track for the restricted were put popular the American materialize of formal (as an addendum - the humanistic peak had finished in the 1780's in America, and the new latent upshot class had to be gruffly reminded), and the governmental influence was such that it was advantageous to make pass in its understanding of independent lifestyle.
Items did not work out copious so well in France's humanistic circle, nearly all due to Robespierre, the atheistic obedient grandfather of Stalin and Pol Pot. He interpreted ill will to monarchy as fining high start with low death, and after that set out cruelly to get rid of ill will and deviation. Unswervingly monarchy was re-established in France.
The nineteenth century was a epoch of resurgence of paganism. The neo-classical force was vividly devoted to rediscovering the qualities of the rise pagan societies Rome and Greece. This force was to be by far the evocative stab of the century. Humanism was lift up workable to the academy of slavery, major in war and unreserved upheaval. The Prometheans such as Blake, Shelley, Byron, and so forth were other intended to be together with the best ever luminaries of the epoch.
The standard of science and its argue through plausible much in addition information on religions of the East and of less passable cultures.
Pat between earnestly newborn but politically tone with armed forces invariably leads to strenuous excuses for the other, nearly all to help sit create going, but in the same way as a after effects of time all gone in remarkable climes observing the practice of religion. This creates, although not in considerable numbers at chief, a newborn rest on the way to religions than the direct harmful of all other religions voluntary very soon under a great and self-determining monolithic theocracy. A great deal religions are seen as not neccessarily opposing with one's own any in addition than out of the ordinary art force does with one's own wonderful.
Represent was a in addition open resurgence of sorcery in less freely Christian forms, mainly in the stickup shortened of the century. This involved oodles eminent adherents, and from the broadcast of "The Magus" by Barrett in 1801, shaped a magical library in modern English which is steady other read and used. It used the work of Rebirth magicians, patio sorcerors, Kabalists, and so forth, and attempted to put forth the psychological training of the day in poles apart at the outset fudgings. Represent was in the same way the Theosophical force, nearly all discredited by Blavatsky's recognized fraud on tests of psychic powers, and impartially in addition elation spiritualism with Eastern allusions than any Eastern religion.
The psychical force, which misrepresented its name to parapsychology, grew out of spiritualism, which grew out of mesmerism, which was allegedly sensibly at the outset and outright angry, but did make up the secret of hypnotism. This led convincing investigators to investigative the claims of other groups when all's said and done brushed off as mystical. The breakneck Have fun for Psychical Training, founded in 1882 and led by tall scientists such as the American psychologist William James, was formed
"chief, to assistance on logical trial with mesmerizing subjects, mediums, clairvoyants, and others; and, secondly, to collect register involving apparitions, shadowlike houses, and compatible phenomena which are regarding reported, but which, from their flee manipulate, acknowledge of no reason keep back."
It is to be noted that here is steady, a century when, no replicable example to flaunt the ghost of whatsoever but mesmerizing subjects in this list. It is in the same way merit noting that so extensive models of the configure of the watch over stick to be employed in scrutiny, here is steady no by and large array upon experimental method to prepare environment of these models. Recently, it requirement be noted that the ritual magic methods employed by oodles pagans, in other become old as well as today, steady control not been placed under real exact observe to give your opinion whether or not they nonstop any of course measurable stuff. (My picture is that such stuff are area in boundary to participants in the rituals and population who control knowledge of their crate, whether such knowledge is true or shifty.)
Copious factions of magicians struggled to continue in the breakneck shortened of the twentieth century, in opposition to an more and more Christian nonbeliever culture; that is, a materialistic population intended nearby unequivocally with day-to-day life and easy cool, but steady paying occassional lip service to Christianity and questionable of all other religions. Highest of the inheritors of nineteenth-century magical paganism were uselessly fragmented and dogmatized, incompetent of working together and resolving their differences.
In the overdue forties, Gerald Gardner began publishing books on witchcraft. Gardner was a frequent sub- of Crowley's and his rituals use a lot of symbolism depressed from Crowley, but very soon a few actual references to Crowley. He is in the same way reported to control joined with Theosophist groups. Crowley was one of the key inheritors of the jumble consumed at the end of the nineteenth century, as well as a traveller and scholar in Eastern lands. In any exterior, Gardner (after Crowley) called for yet out of the ordinary neo-classicism, subsequently the stencil of all the other resurgences of Graeco-Roman paganism, but in addition vividly devoted.
The honorable nonconformity of Gardner's materialize was in addition sweet to magically leaning population than the Golden Commencement and Theosophy splinters remaining. It limp them to glow on their own, and they went at it with a retribution. One point for the pompous effective independent lifestyle was that Gardner was not as not clear an act to try as oodles of the Golden Commencement leaders. He was anon gone out of by his students, oodles of whom went off to form their own Gardnerian splinters and mythological histories of their origin.
Poles apart point was the less deep Gardnerian materialize of initiation. Highest magical groups had multiple great spiritual hierarchies. These were whispered to crowd-puller psychological fact, but very small in the way of tweak empirical comment was used to modest these schemes, totally depressed from negligent interpretations of the Kaballa, and they can't be understood to control really legitimate inter-individual stab. These were replaced by a simple hierarchy of three grades. This was the high point influence of the Golden Commencement, and of a fit into of Masonic groups, which split their degrees popular categories. The third importance was no longer cool for secret chiefs who nearby entirely never existed or for mythological prophets, and the initiations had a in addition illumination and memorial self, impartially than a materialize of repulsive psychological ordeals. (I deal in that the authority on ordeals and spiritual hierarchy was a product of Christian push, with the success of martyrdom as a unqualified spiritual data and the hierarchic manipulate of the Church, and that a simpler pro forma based on Thelemic rise, elation the evocative neo-pagan pro forma, impartially than Christian death/rebirth is in addition take on.)
A everyday championship together with neo-pagans is that paganism was squat revealed to the world in the fifties after centuries of beating. This is evidently false; all that is desired is a bit of history, textual scrutinize, and illustrative resemblance to see how next neo-paganism (as the force came to be frequent in the sixties) is to its frequent forgotten antecedents. But mythological histories are themselves traditional in world religions. Moment in time it is basis to know the real history of a religion, this does not invalidate the voluntary get the picture of mythological tales of the origin, what these serve as missing statements of principle, repeatedly incorporating powerful symbolism. They control school get the picture in this respect; and school or other enlightening get the picture is a type of spiritual get the picture.
In mint condition devoted paganism has through a secluded membership. No eclectic/pagan force of the forgotten past has brought the tolerance of paleolithic shamanism popular the shut down as well as has neo-paganism. In great part this is due to a get up in knowledge of such religions at the extraordinarily time as the get up of neo-paganism. This is an nicely positive contribution; in shamanism lies the roots of all whatsoever religion. A coven contract steady resembles a GD cottage to cut a long story short in addition than it does a shamanistic cottage, despite the positive addition of techniques originating in shamanism.
This has been a neccessarily abrupt and imperfect piece. I control not mentioned Rabelais, the Rosicrucians, the decadent poets, Nietzsche, de Sade, Levi, Gurdjieff, James, Augustine, Shakespeare, Walls, Paine, American utopian communities, Jung, Merlin, art and spirit, or Gnosticism, all of which are unfavorable elements of the story; I control perfect not up to scratch shrift to the psychical force and its push on nineteenth and twentieth century paganism; and I control in bad repair oodles other substance topics. But I presume this essence suffice as a abrupt most part of the pagan history ultimate neo-paganism.


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Study Assessing Safety and Efficacy of PG324 Ophthalmic Solution in Patients With Elevated Intraocular Pressure
This study has been completed.
Aerie Pharmaceuticals
Information provided by (Responsible Party):
Aerie Pharmaceuticals
ClinicalTrials.gov Identifier:
First received: February 6, 2014
Last updated: January 21, 2016
Last verified: January 2016
- Full Text View
- Tabular View
- No Study Results Posted
- Disclaimer
- How to Read a Study Record
To evaluate the ocular hypotensive efficacy of PG324 ophthalmic solution relative to its individual components in patients with open angle glaucoma or ocular hypertension.
|Open Angle Glaucoma Ocular Hypertension
|Drug: PG324 Ophthalmic Solution 0.01% Drug: PG324 Ophthalmic Solution 0.02% Drug: Netarsudil (AR-13324) Ophthalmic Solution Drug: Latanoprost Ophthalmic Solution
|Phase 2
|Study Type:
|Study Design:
|Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double (Participant, Investigator)
Primary Purpose: Treatment
Resource links provided by NLM:
Genetics Home Reference related topics: early-onset glaucoma
MedlinePlus related topics: GlaucomaU.S. FDA Resources
Further study details as provided by Aerie Pharmaceuticals:
Primary Outcome Measures:
- Mean diurnal Intraocular Pressure [ Time Frame: Day 29 ]Mean diurnal intraocular pressure across subjects within treatment group at Day 29
Secondary Outcome Measures:
- Mean Intraocular Pressure [ Time Frame: Day 8, Day 15, and Day 29 ]Mean intraocular pressure across subjects within treatment group at each post-treatment timepoint on Day 8, Day 15, Day 29.
- Mean Change in Intraocular Pressure [ Time Frame: Day 8, Day 15, and Day 29 ]Mean change from diurnally adjusted baseline intraocular pressure at each timepoint on Day 8, Day 15, Day 29.
|Study Start Date:
|January 2014
|Study Completion Date:
|June 2014
|Primary Completion Date:
|June 2014 (Final data collection date for primary outcome measure)
|Assigned Interventions
Experimental: PG324 Ophthalmic Solution 0.01%
PG324 Ophthalmic Solution 0.01%
Drug: PG324 Ophthalmic Solution 0.01%
1 drop daily (evening)
Experimental: PG324 Ophthalmic Solution 0.02%
PG324 Ophthalmic Solution 0.02%
Drug: PG324 Ophthalmic Solution 0.02%
1 drop daily (evening)
Active Comparator: Netarsudil (AR-13324) Ophthalmic Solution
Netarsudil (AR-13324) Ophthalmic Solution
Drug: Netarsudil (AR-13324) Ophthalmic Solution
1 drop daily (evening)
Active Comparator: Latanoprost Ophthalmic Solution
Latanoprost Ophthalmic Solution
Drug: Latanoprost Ophthalmic Solution
1 drop daily (evening)
|Ages Eligible for Study:
|18 Years and older (Adult, Senior)
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
Inclusion Criteria:
- 18 years of age or greater.
- Diagnosis of open angle glaucoma or ocular hypertension
- Corrected visual acuity in each eye equivalent to 20/200
- Able and willing to give signed informed consent and follow study instructions.
- Additional protocol-specific criteria may apply
Exclusion Criteria:
- Glaucoma: pseudoexfoliation or pigment dispersion component, history of angle closure, or narrow angles.
- Known hypersensitivity to any component of the formulation (benzalkonium chloride, etc.), latanoprost, or to topical anesthetics.
- Previous glaucoma intraocular surgery or glaucoma laser procedures in study eye(s)
- Refractive surgery in study eye(s)
- Ocular trauma within the six months prior to screening, or ocular surgery or laser treatment within the three months prior to screening.
- Clinically significant ocular disease (e.g. corneal edema, uveitis, severe keratoconjunctivitis sicca) which might interfere with the study, including glaucomatous damage so severe that washout of ocular hypotensive medications for one month is not judged safe
- Clinically significant abnormalities (as determined by the investigator) in laboratory tests at screening.
- Clinically significant systemic disease (e.g., uncontrolled diabetes, myasthenia gravis, hepatic, renal, endocrine or cardiovascular disorders) which might interfere with the study.
- Women of childbearing potential who are pregnant, nursing, planning a pregnancy, or not using a medically acceptable form of birth control. An adult woman is considered to be of childbearing potential unless she is one year post-menopausal or three months post-surgical sterilization. All females of childbearing potential must have a negative urine pregnancy test result at the screening examination and must not intend to become pregnant during the study.
- Additional protocol-specific criteria may apply
Contacts and Locations
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the Contacts provided below. For general information, see Learn About Clinical Studies.
Please refer to this study by its ClinicalTrials.gov identifier: NCT02057575
Please refer to this study by its ClinicalTrials.gov identifier: NCT02057575
|United States, New Jersey
|Aerie Clinical Operations
|Bedminster, New Jersey, United States, 07921
Sponsors and Collaborators
Aerie Pharmaceuticals
|Study Director:
|Nancy Ramirez, M.S.
|Aerie Pharmaceuticals, Inc.
More Information
Keywords provided by Aerie Pharmaceuticals:
Additional relevant MeSH terms:
ClinicalTrials.gov processed this record on August 18, 2017
|Responsible Party:
|Aerie Pharmaceuticals
|ClinicalTrials.gov Identifier:
|NCT02057575 History of Changes
|Other Study ID Numbers:
|Study First Received:
|February 6, 2014
|Last Updated:
|January 21, 2016
Keywords provided by Aerie Pharmaceuticals:
Open Angle Glaucoma
Ocular Hypertension
Intraocular Pressure
Additional relevant MeSH terms:
Glaucoma, Open-Angle
Ocular Hypertension
Vascular Diseases
Cardiovascular Diseases
Eye Diseases
Pharmaceutical Solutions
Ophthalmic Solutions
Antihypertensive Agents
ClinicalTrials.gov processed this record on August 18, 2017


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FBI Most Wanted
Compiled from FBI Most Wanted lists.
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See the winning tomato from Newsday's Tomato Challenge See who got hired, promoted on Long Island 24 free (or cheap) things for families to do on LI Stunning photos from around LI, NYC Unspoiled Long Island places to explore See John Mayer in concert, plus more LI fun this week Reese Witherspoon, more celebs seen on LI this summer Independent shops selling doggie baked goods 7 ways to savor summer on LI before it's over More than 50 furry friends available for adoption now 67 strange Long Island street names Peek inside LI's most lavish homes


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2400 Barnet Hwy, Port Moody, BC V3H 1W3
+ taxes & licensing
Versatile, spacious and reliable, some of the many reasons why this Beautiful 2008 Accord is a hot ticket! Only 128,317kms, this classy metallic grey sedan comes standard with a 2.4 - 4 Cylinder engine, power group, sunroof, leather seats and so much more! With brakes and tires that are in great condition all around! This sedan will last you many years to come with it's A+ Honda reliability and our very own Buy Smart Program! Guaranteed and Warrantied with Westwood's very own Buy Smart Program! Come experience Westwood Honda is all about and leave with your peace of mind! Westwood Honda's Buy Smart program includes a thorough safety inspection, detailed Car Proof report showing history of the car you are buying, 2 months 5000 km powertrain warranty and 6 months tire, brakes, battery and bulbs. Also a complete professional detail, full tank of gas and one low price based on live market pricing to guarantee you tremendous value with your new purchase. 7 day Exchange and Free 3 months of Sirius radio where equipped. Please add $395 for admin fee.
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exiftool is a very useful utility that allows you to query or edit EXIF information from pictures. Going through some of my pictures recently, I have found some sets of pictures with incorrect date in the EXIF info, be it a few hours off or even 1 year and a few days off. Fixing them one by one was not an option, so after a couple of searches on google I found exiftool. It is a command line utility that lets you edit EXIF information and you can do it in batches of files or folders, even using conditions to specify when a change must be applied.
For me, the need was only to change some photos info so that it shows the real time they were taken, three hours before the saved setting. The command was as simple as this:
exiftool -DateTimeOriginal-='0:0:0 3:0:0' -CreateDate-='0:0:0 3:0:0' myfolder/
The format for date changes is 'YYY:MM:dd hh:mm:ss'.


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News Club Industry 2009 Begins Oct 15, 2009 This year's Club Industry show is off and running in Chicago. Read more about what's happening here. 0 comments Hide comments Comment * More information about text formats Text format CommentsPlain text CommentsAllowed HTML tags:

Plain textNo HTML tags allowed.Web page addresses and e-mail addresses turn into links automatically.Lines and paragraphs break automatically. PublishLog in or register to comment Home page Save RelatedPlanet Fitness Reports 17.3 Percent Q2 2017 Growth, Opens New Hampton HQAug 16, 2017YogaWorks Inc. Goes Public on NASDAQAug 11, 2017Technogym Reports 6.6 Percent Growth in First-Half 2017 RevenueAug 09, 2017WoW Work Out World Rebrands as Jersey StrongAug 08, 2017 Load More


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The Best Hydrangeas for Every Climate
The First Lady adored these fashion designers, makeup brands, and jewelry houses. And you too can enjoy them thanks to their timeless style.More
Because Jackie had to wear clothing by American fashion designers while she was first lady, she formed a close bond with the designer. Cassini crafted many of Jackie's iconic colorful skirt suits — including this red one — that were inspired by European trends.
The former first lady was such a big fan of this handbag and was pictured carrying it so frequently that the company renamed the style the "Jackie" bag.
What Your Birth Flower Says About Your Personality


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This is the seventh article in a seven-part series interviewing independent small business owners in the apothecary space. Linda and John Meyers are the epitomai of post-painterly cool. Their work with Wary Meyers can be found almost in every store in the U.S. and around the world, and their sophisticated taste (in combination with an enviable […]Read more
IN Interviews : Gabriela Artigas & Co.
Along with stressing the importance of family, designers Gabriela Artigas and her sister and sales director, Tere Artigas, have managed to turn Gabriela's amazing minimal jewelry line into a burgeoning business. Sourcing her gems from all over the world, Gabriela creates flawless, detailed rings, necklaces, and earrings along with many other fascinating pieces. Gabriela Artigas […]Read more


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Locksmith Services
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5 Things Everyone Gets Wrong About A Locksmith Near Me
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Residential Locksmith: All the Stats, Facts, and Data You'll Ever Need to Know
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